U.S. patent application number 17/282314 was filed with the patent office on 2021-12-09 for chirally enriched oligomeric compounds.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Stanley T. Crooke, Xue-hai Liang, Michael T. Migawa, Michael Oestergaard, Punit P. Seth, Wen Shen, Eric E. Swayze.
Application Number | 20210380976 17/282314 |
Document ID | / |
Family ID | 1000005827638 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210380976 |
Kind Code |
A1 |
Seth; Punit P. ; et
al. |
December 9, 2021 |
CHIRALLY ENRICHED OLIGOMERIC COMPOUNDS
Abstract
The present disclosure provides oligomeric compounds comprising
a modified oligonucleotide having one or more chirally enriched
phosphorothioate intemucleoside linkages. In certain embodiments,
the modified oligonucleotide decreases expression of target
mRNA.
Inventors: |
Seth; Punit P.; (Carlsbad,
CA) ; Oestergaard; Michael; (Carlsbad, CA) ;
Migawa; Michael T.; (Carlsbad, CA) ; Liang;
Xue-hai; (Del Mar, CA) ; Shen; Wen; (Carlsbad,
CA) ; Crooke; Stanley T.; (Carlsbad, CA) ;
Swayze; Eric E.; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
1000005827638 |
Appl. No.: |
17/282314 |
Filed: |
October 4, 2019 |
PCT Filed: |
October 4, 2019 |
PCT NO: |
PCT/US2019/054667 |
371 Date: |
April 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62742260 |
Oct 5, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/11 20130101; C12N 2310/322 20130101; C12N 2310/341
20130101; C12N 2310/315 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. A chirally enriched population of oligomeric compounds
comprising modified oligonucleotides, wherein the modified
oligonucleotides consist of 12-23 linked nucleosides, wherein the
modified oligonucleotide comprises a gapmer consisting of a
5'-region, a central region, and a 3'-region wherein: the 5'-region
consists of 1-5 linked modified nucleosides, wherein each
nucleoside of the 5'-region comprises a 2'-modified furanosyl sugar
moiety; the 3'-region consists of 1-5 linked modified nucleosides,
wherein each nucleoside of the 3'-region comprises a 2'-modified
furanosyl sugar moiety; the central region consists of 7-10 linked
nucleosides, where each nucleoside of the central region comprises
a 2'-.beta.-D-deoxyribosyl sugar moiety; and wherein the central
region has at least one phosphorothioate internucleoside linkage
that is chirally enriched in the (Sp) configuration or the (Rp)
configuration.
2. A chirally enriched population of oligomeric compounds, wherein
the oligomeric compounds comprise a modified oligonucleotide
consisting of 12-23 linked nucleosides, wherein the modified
oligonucleotide is a gapmer consisting of a 5'-region, a central
region, and a 3'-region wherein: the 5'-region consists of 1-5
linked modified nucleosides, wherein each nucleoside of the
5'-region comprises a 2'-modified furanosyl sugar moiety; the
3'-region consists of 1-5 linked modified nucleosides, wherein each
nucleoside of the 3'-region comprises a 2'-modified furanosyl sugar
moiety; the central region consists of 7-10 linked nucleosides,
where each nucleoside of the central region comprises a
2'-.beta.-D-deoxyribosyl sugar moiety; wherein the central region
has at least one phosphorothioate internucleoside linkage; and
wherein a percentage of oligomeric compounds within the population
that contain a stereochemical configuration at the phosphorothioate
internucleoside linkage is greater than an expected percentage of
oligomeric compounds expected to contain the same particular
stereochemical configuration at the phosphorothioate
internucleoside linkage within the population if the stereochemical
configuration at the phosphorothioate internucleoside linkage was
stereorandom.
3. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein each central region internucleoside linkage
is selected from among a phosphodiester internucleoside linkage and
a phosphorothioate internucleoside linkage.
4. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 2 phosphorothioate
internucleoside linkages.
5. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 3 phosphorothioate
internucleoside linkages.
6. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 4 phosphorothioate
internucleoside linkages.
7. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 5 phosphorothioate
internucleoside linkages.
8. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 6 phosphorothioate
internucleoside linkages.
9. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 7 phosphorothioate
internucleoside linkages.
10. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 8 phosphorothioate
internucleoside linkages.
11. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 9 phosphorothioate
internucleoside linkages.
12. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 10 phosphorothioate
internucleoside linkages.
13. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region has 11 phosphorothioate
internucleoside linkages.
14. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein each central region internucleoside linkage
is a phosphorothioate internucleoside linkage.
15. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein each phosphorothioate internucleoside linkage
has the (Sp) configuration.
16. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein each phosphorothioate internucleoside linkage
has the (Rp) configuration.
17. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein 1 central region internucleoside linkage has
the (Rp) configuration.
18. The chirally enriched population of oligomeric compounds of any
of claims 1-14, wherein 2 central region internucleoside linkages
have the (Rp) configuration.
19. The chirally enriched population of oligomeric compounds of
claim 18, wherein the 2 central region internucleoside linkages
having the (Rp) configuration are adjacent.
20. The chirally enriched population of oligomeric compounds of
claim 1 or 2, wherein the central region consists of 7 linked
nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7; wherein each N.sub.d is a
2'-.beta.-D-deoxyribosyl sugar moiety and each of L1, L2, L3, L4,
L5, L6, and L7 is a phosphorothioate internucleoside linkage; and
wherein at least one of L1, L2, L3, L4, L5, L6, and L7 has the (Sp)
configuration or the (Rp) configuration.
21. The chirally enriched population of oligomeric compounds of
claim 20, wherein L1 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L2, L3, L4, L5, L6,
and L7 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
22. The chirally enriched population of oligomeric compounds of
claim 20, wherein L.sub.2 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L3, L4,
L5, L6, and L7 is a phosphorothioate internucleoside linkage having
the (Sp) configuration.
23. The chirally enriched population of oligomeric compounds of
claim 20, wherein L3 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L4, L5, L6,
and L7 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
24. The chirally enriched population of oligomeric compounds of
claim 20, wherein L4 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L5, L6,
and L7 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
25. The chirally enriched population of oligomeric compounds of
claim 20, wherein L5 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L6,
and L7 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
26. The chirally enriched population of oligomeric compounds of
claim 20, wherein L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
and L7 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
27. The chirally enriched population of oligomeric compounds of
claim 20, wherein L7 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
and L6 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
28. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L1 and L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L3, L4, L5, L6, and L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
29. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L.sub.2 and L3 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L4, L5, L6, and L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
30. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L3 and L4 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L5, L6, and L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
31. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L4 and L5 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L6, and L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
32. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L5 and L6 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, and L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
33. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L6 and L7 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, and L5 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
34. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L1 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L.sub.2 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L3, L4, L5, L6 and L7 is a
stereorandom phosphorothioate internucleoside linkage.
35. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L3 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L4, L5, L6 and L7 is a
stereorandom phosphorothioate internucleoside linkage.
36. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L3 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L4 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L5, L6 and L7 is a
stereorandom phosphorothioate internucleoside linkage.
37. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L4 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L5 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L6 and L7 is a
stereorandom phosphorothioate internucleoside linkage.
38. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L5 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L6 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4 and L7 is a
stereorandom phosphorothioate internucleoside linkage.
39. The chirally enriched population of oligomeric compounds of
claim 20, wherein each L6 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4 and L5 is a
stereorandom phosphorothioate internucleoside linkage.
40. The oligomeric compound of claim 1 or 2, wherein the central
region consists of 8 linked nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7(N.sub.d).sub.L8; wherein
each N.sub.d is a 2'-.beta.-D-deoxyribosyl sugar moiety and each of
L1, L2, L3, L4, L5, L6, L7, and L8 is a phosphorothioate
internucleoside linkage; and wherein at least one of L1, L2, L3,
L4, L5, L6, L7 and L8 has the (Sp) configuration or the (Rp)
configuration.
41. The oligomeric compound of claim 1 or 2, wherein the central
region consists of 9 linked nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7(N.sub.d).sub.L8(N.sub.d).sub.L9;
wherein each N.sub.d is a 2'-.beta.-D-deoxyribosyl sugar moiety and
each of L1, L2, L3, L4, L5, L6, L7, L8, and L9 is a
phosphorothioate internucleoside linkage; and wherein at least one
of L1, L2, L3, L4, L5, L6, L7, L8 and L9 has the (Sp) configuration
or the (Rp) configuration.
42. The chirally enriched population of oligomeric compounds of
claim 41, wherein L1 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L2, L3, L4, L5, L6,
L7 and L8 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
43. The chirally enriched population of oligomeric compounds of
claim 41, wherein L.sub.2 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L3, L4,
L5, L6, L7 and L8 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
44. The chirally enriched population of oligomeric compounds of
claim 41, wherein L3 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L4, L5, L6,
L7 and L8 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
45. The chirally enriched population of oligomeric compounds of
claim 41, wherein L4 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L4, L5, L6,
L7 and L8 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
46. The chirally enriched population of oligomeric compounds of
claim 41, wherein L5 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L6,
L7 and L8 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
47. The chirally enriched population of oligomeric compounds of
claim 41, wherein L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L7 and L8 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
48. The chirally enriched population of oligomeric compounds of
claim 41, wherein L7 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6 and L8 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
49. The chirally enriched population of oligomeric compounds of
claim 41, wherein L8 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6 and L7 is a phosphorothioate internucleoside linkage having the
(Sp) configuration.
50. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L1 and L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L3, L4, L5, L6 and L8 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
51. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L.sub.2 and L3 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L4, L5, L6 and L8 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
52. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L3 and L4 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L5, L6 and L8 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
53. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L4 and L5 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L6 and L8 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
54. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L5 and L6 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L6 and L8 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
55. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L6 and L7 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L5 and L8 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
56. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L7 and L8 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L5 and L6 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
57. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L1 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L.sub.2 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L3, L4, L5, L6, L7 and L8 is a
stereorandom phosphorothioate internucleoside linkage.
58. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L3 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L4, L5, L6, L7 and L8 is a
stereorandom phosphorothioate internucleoside linkage.
59. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L3 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L4 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L5, L6, L7 and L8 is a
stereorandom phosphorothioate internucleoside linkage.
60. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L4 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L5 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L6, L7 and L8 is a
stereorandom phosphorothioate internucleoside linkage.
61. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L5 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L6 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L7 and L8 is a
stereorandom phosphorothioate internucleoside linkage.
62. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L6 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5 and L8 is a
stereorandom phosphorothioate internucleoside linkage.
63. The chirally enriched population of oligomeric compounds of
claim 41, wherein each L7 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5 and L6 is a
stereorandom phosphorothioate internucleoside linkage.
64. The oligomeric compound of claim 1 or 2, wherein the central
region consists of 9 linked nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7(N.sub.d).sub.L8(N.sub.d).sub.L9;
wherein each N.sub.d is a 2'-.beta.-D-deoxyribosyl sugar moiety and
each of L1, L2, L3, L4, L5, L6, L7, L8, and L9 is a
phosphorothioate internucleoside linkage; and wherein at least one
of L1, L2, L3, L4, L5, L6, L7, L8 and L9 has the (Sp) configuration
or the (Rp) configuration.
65. The chirally enriched population of oligomeric compounds of
claim 51 or 64, wherein L1 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L2, L3, L4,
L5, L6, L7, L8 and L9 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
66. The chirally enriched population of oligomeric compounds of
claim 64, wherein L.sub.2 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L3, L4,
L5, L6, L7, L8 and L9 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
67. The chirally enriched population of oligomeric compounds of
claim 64, wherein L3 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L4, L5, L6,
L7, L8 and L9 is a phosphorothioate internucleoside linkage having
the (Sp) configuration.
68. The chirally enriched population of oligomeric compounds of
claim 64, wherein L4 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L5, L6,
L7, L8 and L9 is a phosphorothioate internucleoside linkage having
the (Sp) configuration.
69. The chirally enriched population of oligomeric compounds of
claim 64, wherein L5 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L6,
L7, L8 and L9 is a phosphorothioate internucleoside linkage having
the (Sp) configuration.
70. The chirally enriched population of oligomeric compounds of
claim 64, wherein L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L7, L8 and L9 is a phosphorothioate internucleoside linkage having
the (Sp) configuration.
71. The chirally enriched population of oligomeric compounds of
claim 64, wherein L7 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L8 and L9 is a phosphorothioate internucleoside linkage having
the (Sp) configuration.
72. The chirally enriched population of oligomeric compounds of
claim 64, wherein L8 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7 and L9 is a phosphorothioate internucleoside linkage having
the (Sp) configuration.
73. The chirally enriched population of oligomeric compounds of
claim 64, wherein L9 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7 and L8 is a phosphorothioate internucleoside linkage having
the (Sp) configuration.
74. The chirally enriched population of oligomeric compounds of
claim 64, wherein each of L1 and L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L3, L4, L5, L6, L7, L8 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
75. The chirally enriched population of oligomeric compounds of
claim 64, wherein each of L.sub.2 and L3 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L4, L5, L6, L7, L8 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
76. The chirally enriched population of oligomeric compounds of
claim 64, wherein each of L3 and L4 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L5, L6, L7, L8 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
77. The chirally enriched population of oligomeric compounds of
claim 64, wherein each of L4 and L5 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L6, L7, L8 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
78. The chirally enriched population of oligomeric compounds of
claim 64, wherein each of L5 and L6 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L7, L8 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
79. The chirally enriched population of oligomeric compounds of
claim 64, wherein each of L6 and L7 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L8 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
80. The chirally enriched population of oligomeric compounds of
claim 64, wherein each of L7 and L8 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
81. The chirally enriched population of oligomeric compounds of
claim 64, wherein each of L8 and L9 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6 and L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
82. The chirally enriched population of oligomeric compounds of
claim 64, wherein each L1 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L.sub.2 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L3, L4, L5, L6, L7, L8 and L9 is a
stereorandom phosphorothioate internucleoside linkage.
83. The chirally enriched population of oligomeric compounds of
claim 64, wherein each L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L3 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L4, L5, L6, L7, L8 and L9 is a
stereorandom phosphorothioate internucleoside linkage.
84. The chirally enriched population of oligomeric compounds of
claim 64, wherein each L3 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L4 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L5, L6, L7, L8 and L9 is a
stereorandom phosphorothioate internucleoside linkage.
85. The chirally enriched population of oligomeric compounds of
claim 64, wherein each L4 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L5 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L6, L7, L8 and L9 is a
stereorandom phosphorothioate internucleoside linkage.
86. The chirally enriched population of oligomeric compounds of
claim 64, wherein each L5 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L6 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L7, L8 and L9 is a
stereorandom phosphorothioate internucleoside linkage.
87. The chirally enriched population of oligomeric compounds of
claim 64, wherein each L6 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L8 and L9 is a
stereorandom phosphorothioate internucleoside linkage.
88. The chirally enriched population of oligomeric compounds of
claim 64, wherein each L7 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6 and L9 is a
stereorandom phosphorothioate internucleoside linkage.
89. The chirally enriched population of oligomeric compounds of
claim 64, wherein each L8 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L9 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6 and L7 is a
stereorandom phosphorothioate internucleoside linkage.
90. The oligomeric compound of claim 1 or 2, wherein the central
region consists of 10 linked nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7(N.sub.d).sub.L8(N.sub.d).sub.L9(N.-
sub.d).sub.L10; wherein each N.sub.d is a 2'-.beta.-D-deoxyribosyl
sugar moiety and each of L1, L2, L3, L4, L5, L6, L7, L8, L9, and
L10 is a phosphorothioate internucleoside linkage; and wherein at
least one of L1, L2, L3, L4, L5, L6, L7, L8, L9 and L10 has the
(Sp) configuration or the (Rp) configuration.
91. The chirally enriched population of oligomeric compounds of
claim 69 or 90, wherein L1 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L2, L3, L4,
L5, L6, L7, L8, L9 and L10 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
92. The chirally enriched population of oligomeric compounds of
claim 90, wherein L.sub.2 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L3, L4,
L5, L6, L7, L8, L9 and L10 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
93. The chirally enriched population of oligomeric compounds of
claim 90, wherein L3 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L4, L5, L6,
L7, L8, L9 and L10 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
94. The chirally enriched population of oligomeric compounds of
claim 90, wherein L4 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L5, L6,
L7, L8, L9 and L10 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
95. The chirally enriched population of oligomeric compounds of
claim 90, wherein L5 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L6,
L7, L8, L9 and L10 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
96. The chirally enriched population of oligomeric compounds of
claim 90, wherein L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L7, L8, L9 and L10 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
97. The chirally enriched population of oligomeric compounds of
claim 90, wherein L7 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L8, L9 and L10 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
98. The chirally enriched population of oligomeric compounds of
claim 90, wherein L8 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L9 and L10 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
99. The chirally enriched population of oligomeric compounds of
claim 90, wherein L9 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L8 and L10 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
100. The chirally enriched population of oligomeric compounds of
claim 90, wherein L10 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L8 and L9 is a phosphorothioate internucleoside linkage
having the (Sp) configuration.
101. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L1 and L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L3, L4, L5, L6, L7, L8, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
102. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L.sub.2 and L3 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L4, L5, L6, L7, L8, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
103. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L3 and L4 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L5, L6, L7, L8, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
104. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L4 and L5 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L6, L7, L8, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
105. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L5 and L6 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L7, L8, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
106. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L6 and L7 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L8, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
107. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L7 and L8 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
108. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L8 and L9 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L7 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
109. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L9 and L10 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L7 and L8 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
110. The chirally enriched population of oligomeric compounds of
claim 90, wherein each of L3, L6, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L4, L5, L7, and L8 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
111. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L1 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L.sub.2 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L3, L4, L5, L6, L7, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage.
112. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L3 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L4, L5, L6, L7, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage.
113. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L3 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L4 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L5, L6, L7, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage.
114. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L4 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L5 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L6, L7, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage.
115. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L5 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L6 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L7, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage.
116. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L6 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage.
117. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L7 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage.
118. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L8 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L9 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L7 and L10
is a stereorandom phosphorothioate internucleoside linkage.
119. The chirally enriched population of oligomeric compounds of
claim 90, wherein each L9 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L10 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L7 and L8
is a stereorandom phosphorothioate internucleoside linkage.
120. The oligomeric compound of claim 1 or 2, wherein the central
region consists of 11 linked nucleosides and has the formula:
(Nd)L1(Nd)L2(Nd)L3(Nd)L4(Nd)L5(Nd)L6(Nd)L7(Nd)L8(Nd)L9(Nd)L10(Nd)L11;
wherein, each N.sub.d is a 2'-.beta.-D-deoxyribosyl sugar moiety
and each of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage; and wherein at least one
of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10 and L11 has the (Sp)
configuration or the (Rp) configuration.
121. The chirally enriched population of oligomeric compounds of
claim 120, wherein L1 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L2, L3, L4, L5, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
122. The chirally enriched population of oligomeric compounds of
claim 120, wherein L.sub.2 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L3, L4,
L5, L6, L7, L8, L9, L10 and L11 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
123. The chirally enriched population of oligomeric compounds of
claim 120, wherein L3 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L4, L5, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
124. The chirally enriched population of oligomeric compounds of
claim 120, wherein L4 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L5, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
125. The chirally enriched population of oligomeric compounds of
claim 120, wherein L5 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
126. The chirally enriched population of oligomeric compounds of
claim 120, wherein L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
127. The chirally enriched population of oligomeric compounds of
claim 120, wherein L7 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
128. The chirally enriched population of oligomeric compounds of
claim 120, wherein L8 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
129. The chirally enriched population of oligomeric compounds of
claim 120, wherein L9 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L8, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration.
130. The chirally enriched population of oligomeric compounds of
claim 120, wherein L10 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L2, L3,
L4, L5, L6, L7, L8, L9 and L11 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
131. The chirally enriched population of oligomeric compounds of
claim 120, wherein L11 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L2, L3,
L4, L5, L6, L7, L8, L9 and L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
132. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L1 and L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L3, L4, L5, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
133. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L.sub.2 and L3 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L4, L5, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
134. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L.sub.2 and L3 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L4, L5, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
135. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L3 and L4 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L5, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
136. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L4 and L5 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
137. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L5 and L6 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
138. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L6 and L7 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
139. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L7 and L8 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
140. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L8 and L9 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L7, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
141. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L9 and L10 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L7, L9 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
142. The chirally enriched population of oligomeric compounds of
claim 120, wherein each of L10 and L11 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L7, L8 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration.
143. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L1 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L.sub.2 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L3, L4, L5, L6, L7, L8, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside
linkage.
144. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L.sub.2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L3 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L4, L5, L6, L7, L8, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside
linkage.
145. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L3 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L4 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L5, L6, L7, L8, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside
linkage.
146. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L4 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L5 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L6, L7, L8, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside
linkage.
147. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L5 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L6 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and wherein each of L1, L2, L3, L4, L7, L8, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside
linkage.
148. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L6 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L8, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside
linkage.
149. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L7 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside
linkage.
150. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L8 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L9 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L7, L10
and L11 is a stereorandom phosphorothioate internucleoside
linkage.
151. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L9 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L10 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L7, L8 and
L11 is a stereorandom phosphorothioate internucleoside linkage.
152. The chirally enriched population of oligomeric compounds of
claim 120, wherein each L10 is a phosphorothioate internucleoside
linkage having the (Rp) configuration, each L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L7, L8 and
L9 is a stereorandom phosphorothioate internucleoside linkage.
153. The chirally enriched population of oligomeric compounds of
any of claims 1-152, wherein the 5'-region consists of 2-4 linked
nucleosides.
154. The chirally enriched population of oligomeric compounds of
any of claims 1-152, wherein the 5'-region consists of 1
nucleoside.
155. The chirally enriched population of oligomeric compounds of
any of claims 1-152, wherein the 5'-region consists of 2 linked
nucleosides.
156. The chirally enriched population of oligomeric compounds of
any of claims 1-152, wherein the 5'-region consists of 3 linked
nucleosides.
157. The chirally enriched population of oligomeric compounds of
any of claims 1-152, wherein the 5'-region consists of 4 linked
nucleosides.
158. The chirally enriched population of oligomeric compounds of
any of claims 1-152, wherein the 5'-region consists of 5 linked
nucleosides.
159. The chirally enriched population of oligomeric compounds of
any of claims 1-158, wherein each nucleoside of the 5'-region
comprises a 2'-modified ribosyl sugar moiety.
160. The chirally enriched population of oligomeric compounds of
any of claims 1-159, wherein at least one nucleoside of the
5'-region comprises a bicyclic furanosyl sugar moiety.
161. The chirally enriched population of oligomeric compounds of
any of claims 1-160, wherein each nucleoside of the 5'-region
comprises a bicyclic furanosyl sugar moiety.
162. The chirally enriched population of oligomeric compounds of
any of claims 1-160 wherein at least one nucleoside of the
5'-region comprises a non-bicyclic sugar moiety.
163. The chirally enriched population of oligomeric compounds of
claim 162, wherein the non-bicyclic sugar moiety of the at least
one nucleoside of the 5'-region is a 2'-substituted ribosyl sugar
moiety.
164. The chirally enriched population of oligomeric compounds of
any of claim 1-159 or 162-163, wherein each nucleoside of the
5'-region comprises a non-bicyclic sugar moiety.
165. The chirally enriched population of oligomeric compounds of
claim 164, wherein each nucleoside of the 5'-region comprises a
2'-substituted ribosyl sugar moiety.
166. The chirally enriched population of oligomeric compounds of
any of claims 1-159, wherein each nucleoside of the 5'-region
comprises a 2'-modified furanosyl sugar moiety independently
selected from a bicyclic sugar moiety and a non-bicyclic,
2'-substituted ribosyl sugar moiety.
167. The chirally enriched population of oligomeric compounds of
any of claim 160-163 or 166, wherein each bicyclic sugar moiety of
the 5'-region is selected from among cEt, LNA, and ENA.
168. The chirally enriched population of oligomeric compounds of
any of claims 162-166, wherein each non-bicyclic sugar moiety of
the 5'-region has a 2'-substituent selected from among 2'-MOE,
2'-OMe, and 2'-NMA.
169. The chirally enriched population of oligomeric compounds of
any of claims 1-168, wherein none of the nucleosides of the
5'-region comprise a sugar moiety having a 2'-F substituent.
170. The chirally enriched population of oligomeric compounds of
any of claims 1-169, wherein each nucleobase of the 5'-region is
independently selected from among thymine, uracil, guanine,
cytosine, 5-methylcytosine, and adenine.
171. The chirally enriched population of oligomeric compounds of
any of claims 1-170, wherein each internucleoside linkage of the
5'-region is selected from among phosphodiester and
phosphorothioate internucleoside linkages.
172. The chirally enriched population of oligomeric compounds of
any of claims 1-171, wherein the 3'-region consists of 2-4 linked
nucleosides.
173. The chirally enriched population of oligomeric compounds of
any of claims 1-171, wherein the 3'-region consists of 1
nucleoside.
174. The chirally enriched population of oligomeric compounds of
any of claims 1-171, wherein the 3'-region consists of 2 linked
nucleosides.
175. The chirally enriched population of oligomeric compounds of
any of claims 1-171, wherein the 3'-region consists of 3 linked
nucleosides.
176. The chirally enriched population of oligomeric compounds of
any of claims 1-170, wherein the 3'-region consists of 4 linked
nucleosides.
177. The chirally enriched population of oligomeric compounds of
any of claims 1-171, wherein the 3'-region consists of 5 linked
nucleosides.
178. The chirally enriched population of oligomeric compounds of
any of claims 1-177, wherein each nucleoside of the 3'-region
comprises a 2'-modified ribosyl sugar moiety.
179. The chirally enriched population of oligomeric compounds of
any of claims 1-178, wherein at least one nucleoside of the
3'-region comprises a bicyclic furanosyl sugar moiety.
180. The chirally enriched population of oligomeric compounds of
any of claims 1-179, wherein each nucleoside of the 3'-region
comprises a bicyclic furanosyl sugar moiety.
181. The chirally enriched population of oligomeric compounds of
any of claims 1-179, wherein at least one nucleoside of the
3'-region comprises a non-bicyclic sugar moiety.
182. The chirally enriched population of oligomeric compounds of
claim 1-181, wherein the non-bicyclic sugar moiety of the at least
one nucleoside of the 3'-region is a 2'-substituted ribosyl sugar
moiety.
183. The chirally enriched population of oligomeric compounds of
any of claim 1-178 or 181-182, wherein each nucleoside of the
3'-region comprises a non-bicyclic sugar moiety.
184. The chirally enriched population of oligomeric compounds of
claim 183, wherein each nucleoside of the 3'-region comprises a
2'-substituted ribosyl sugar moiety.
185. The chirally enriched population of oligomeric compounds of
any of claims 1-178, wherein each nucleoside of the 3'-region
comprises a 2'-modified furanosyl sugar moiety independently
selected from a bicyclic sugar moiety and a non-bicyclic,
2'-substituted ribosyl sugar moiety.
186. The chirally enriched population of oligomeric compounds of
any of claim 179-182 or 185, wherein each bicyclic sugar moiety of
the 3'-region is selected from among cEt, LNA, and ENA.
187. The chirally enriched population of oligomeric compounds of
any of claims 181-185, wherein each non-bicyclic sugar moiety of
the 3'-region has a 2'-substituent selected from among 2'-MOE,
2'-OMe, and 2'-NMA.
188. The chirally enriched population of oligomeric compounds of
any of claims 1-187, wherein none of the nucleosides of the
3'-region comprise a sugar moiety having a 2'-F substituent.
189. A pharmaceutical composition comprising, the chirally enriched
population of oligomeric compounds of any of claims 1-187.
190. A method comprising, contacting a cell with the chirally
enriched population of oligomeric compounds of any of claims
1-187.
191. A method of modulating the amount or activity of a target
nucleic acid in a cell, comprising contacting a cell with the
chirally enriched population of compounds of any of claims 1-187.
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled CORE0151WOSEQ_ST25.txt created Oct. 3, 2019 which is
20 kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD
[0002] The present disclosure provides oligomeric compounds
comprising a modified oligonucleotide having one or more chirally
enriched phosphorothioate internucleoside linkages. In certain
embodiments, the modified oligonucleotide decreases expression of
target mRNA.
BACKGROUND
[0003] The principle behind antisense technology is that an
antisense compound hybridizes to a target nucleic acid and
modulates the amount, activity, and/or function of the target
nucleic acid. In one example, target RNA function is modulated via
degradation by RNase H upon hybridization with a DNA-like antisense
compound. Another example of modulation of gene expression by
target degradation is RNA interference (RNAi). RNAi refers to
antisense-mediated gene silencing through a mechanism that utilizes
the RNA-induced silencing complex (RISC). Regardless of the
specific mechanism, sequence specificity makes antisense compounds
attractive as tools for target validation and gene
functionalization, as well as therapeutics to selectively modulate
the expression of genes involved in the pathogenesis of
disease.
[0004] Antisense technology is an effective means for modulating
the expression of one or more specific gene products and can
therefore prove to be uniquely useful in a number of therapeutic,
diagnostic, and research applications. Chemically modified
nucleosides may be incorporated into antisense compounds to enhance
one or more properties, such as nuclease resistance,
pharmacokinetics, or affinity for a target nucleic acid.
SUMMARY
[0005] The present disclosure provides chirally enriched
populations of oligomeric compounds comprising modified
oligonucleotides having a region of 2'-.beta.-D-deoxyribosyl sugar
moieties linked through phosphorothioate internucleoside linking
groups chirally enriched in the (Sp) configuration or the (Rp)
configuration. Controlling the chirality of one or more
phosphorothioate internucleoside linkages can alter the properties
of a modified oligonucleotide. For example, site specific
introduction of (Sp) or (Rp) phosphorothioate internucleoside
linkages into the central region of a modified oligonucleotide can
enhance the properties of the modified oligonucleotide. For
example, in certain embodiments, site specific introduction of (Sp)
or (Rp) phosphorothioate internucleoside linkages into the central
region of a modified oligonucleotide can mitigate toxicity of an
otherwise cytotoxic modified oligonucleotide having a stereorandom
configuration at the phosphorothioate internucleoside linkages.
[0006] Site specific introduction of (Sp) or (Rp) phosphorothioate
internucleoside linkages into the central region of a modified
oligonucleotide can alter the protein binding properties of the
modified oligonucleotide. In certain embodiments, site specific
introduction of (Sp) or (Rp) phosphorothioate internucleoside
linkages into the central region of a modified oligonucleotide can
alter the cleavage pattern of RNase H1 when the modified
oligonucleotide binds to a target nucleic acid. In certain
embodiments, site specific introduction of
[0007] (Sp) or (Rp) phosphorothioate internucleoside linkages into
the central region of a modified oligonucleotide can increase the
therapeutic index relative to an otherwise identifical modified
oligonucleotide having a stereorandom configuration at the
phosphorothioate internucleoside linkages.
DETAILED DESCRIPTION
[0008] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the embodiments, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting.
[0009] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, treatises, and GenBank and NCBI
reference sequence records are hereby expressly incorporated by
reference for the portions of the document discussed herein, as
well as in their entirety.
[0010] It is understood that the sequence set forth in each SEQ ID
NO contained herein is independent of any modification to a sugar
moiety, an internucleoside linkage, or a nucleobase. As such,
compounds defined by a SEQ ID NO may comprise, independently, one
or more modifications to a sugar moiety, an internucleoside
linkage, or a nucleobase.
[0011] As used herein, "2'-deoxyfuranosyl sugar moiety" or
"2'-deoxyfuranosyl sugar" means a furanosyl sugar moiety having two
hydrogens at the 2'-position. 2'-deoxyfuranosyl sugar moieties may
be unmodified or modified and may be substituted at positions other
than the 2'-position or unsubstituted. A .beta.-D-2'-deoxyribosyl
sugar moiety or 2'-.beta.-D-deoxyribosyl sugar moiety in the
context of an oligonucleotide is an unsubstituted, unmodified
2'-deoxyfuranosyl and is found in naturally occurring
deoxyribonucleic acids (DNA).
[0012] As used herein, "2'-modified" in reference to a furanosyl
sugar moiety or nucleoside comprising a furanosyl sugar moiety
means the furanosyl sugar moiety comprises a substituent other than
H or OH at the 2'-position of the furanosyl sugar moiety.
2'-modified furanosyl sugar moieties include non-bicyclic and
bicyclic sugar moieties and may comprise, but are not required to
comprise, additional substituents at other positions of the
furanosyl sugar moiety.
[0013] As used herein, "2'-substituted" in reference to a furanosyl
sugar moiety or nucleoside comprising a furanosyl sugar moiety
means the furanosyl sugar moiety or nucleoside comprising the
furanosyl sugar moiety comprises a substituent other than H or OH
at the 2'-position and is a non-bicyclic furanosyl sugar moiety.
2'-substituted furanosyl sugar moieties do not comprise additional
substituents at other positions of the furanosyl sugar moiety other
than a nucleobase and/or internucleoside linkage(s) when in the
context of an oligonucleotide.
[0014] As used herein, "administration" or "administering" refers
to routes of introducing a compound or composition provided herein
to a subject to perform its intended function. Examples of routes
of administration that can be used include, but are not limited to,
administration by inhalation, subcutaneous injection, intrathecal
injection, and oral administration.
[0015] As used herein, "administered concomitantly" or
"co-administration" means administration of two or more compounds
in any manner in which the pharmacological effects of both are
manifest in the patient. Concomitant administration does not
require that both compounds be administered in a single
pharmaceutical composition, in the same dosage form, by the same
route of administration, or at the same time. The effects of both
compounds need not manifest themselves at the same time. The
effects need only be overlapping for a period of time and need not
be coextensive. Concomitant administration or co-administration
encompasses administration in parallel, sequentially, separate, or
simultaneous administration.
[0016] As used herein, "animal" refers to a human or non-human
animal, including, but not limited to, mice, rats, rabbits, dogs,
cats, pigs, and non-human primates, including, but not limited to,
monkeys and chimpanzees.
[0017] As used herein, "antisense activity" means any detectable
and/or measurable change attributable to the hybridization of an
antisense compound to its target nucleic acid. In certain
embodiments, antisense activity is a decrease in the amount or
expression of a target nucleic acid or protein encoded by such
target nucleic acid compared to target nucleic acid levels or
target protein levels in the absence of the antisense compound.
[0018] As used herein, "antisense compound" means a compound
comprising an antisense oligonucleotide and optionally one or more
additional features, such as a conjugate group or terminal
group.
[0019] As used herein, "antisense oligonucleotide" means an
oligonucleotide having a nucleobase sequence that is at least
partially complementary to a target nucleic acid.
[0020] As used herein, "ameliorate" in reference to a treatment
means improvement in at least one symptom relative to the same
symptom in the absence of the treatment. In certain embodiments,
amelioration is the reduction in the severity or frequency of a
symptom or the delayed onset or slowing of progression in the
severity or frequency of a symptom.
[0021] As used herein, "bicyclic nucleoside" or "BNA" means a
nucleoside comprising a bicyclic sugar moiety. As used herein,
"bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar
moiety comprising two rings, wherein the second ring is formed via
a bridge connecting two of the atoms in the first ring thereby
forming a bicyclic structure. In certain embodiments, the first
ring of the bicyclic sugar moiety is a furanosyl moiety, and the
bicyclic sugar moiety is a modified furanosyl sugar moiety. In
certain embodiments, the bicyclic sugar moiety does not comprise a
furanosyl moiety. As used herein, "cEt" or "constrained ethyl"
means a bicyclic sugar moiety, wherein the first ring of the
bicyclic sugar moiety is a ribosyl sugar moiety, the second ring of
the bicyclic sugar is formed via a bridge connecting the 4'-carbon
and the 2'-carbon, the bridge has the formula 4'-CH(CH3)-O-2', and
the methyl group of the bridge is in the S configuration. A cEt
bicyclic sugar moiety is in the (.beta.-D configuration.
[0022] As used herein, "coding region" in the context of an RNA
means the portion of the RNA that is translated into an amino acid
sequence. The coding region of an mRNA excludes the 5'-untranslated
region and the 3'-untranslated region.
[0023] As used herein, "chirally enriched population" means a
plurality of molecules of identical molecular formula, wherein the
number or percentage of molecules within the population that
contain a particular stereochemical configuration at a particular
chiral center is greater than the number or percentage of molecules
expected to contain the same particular stereochemical
configuration at the same particular chiral center within the
population if the particular chiral center were stereorandom.
Chirally enriched populations of molecules having multiple chiral
centers within each molecule may contain one or more sterorandom
chiral centers. In certain embodiments, the molecules are modified
oligonucleotides. In certain embodiments, the molecules are
compounds comprising modified oligonucleotides.
[0024] As used herein, "complementary" in reference to an
oligonucleotide or a region of an oligonucleotide means that at
least 70% of the nucleobases of the entire oligonucleotide or the
region of the oligonucleotide, respectively, and the nucleobases of
another nucleic acid or one or more regions thereof are capable of
hydrogen bonding with one another when the nucleobase sequence of
the oligonucleotide and the other nucleic acid are aligned in
opposing directions. Complementary nucleobases are nucleobase pairs
that are capable of forming hydrogen bonds with one another.
Complementary nucleobase pairs include adenine (A) and thymine (T),
adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methyl
cytosine (.sup.mC) and guanine (G). Complementary oligonucleotides
and/or nucleic acids need not have nucleobase complementarity at
each nucleoside. Rather, some mismatches are tolerated. As used
herein, "fully complementary" or "100% complementary" in reference
to oligonucleotides means that such oligonucleotides are
complementary to another oligonucleotide or nucleic acid at each
nucleoside of the oligonucleotide.
[0025] As used herein, "conjugate group" means a group of atoms
that is directly or indirectly attached to an oligonucleotide.
Conjugate groups may comprise a conjugate moiety and a conjugate
linker that attaches the conjugate moiety to the
oligonucleotide.
[0026] As used herein, "conjugate linker" means a group of atoms
comprising at least one bond that connects a conjugate moiety to an
oligonucleotide.
[0027] As used herein, "conjugate moiety" means a group of atoms
that is attached to an oligonucleotide via a conjugate linker.
[0028] As used herein, "contiguous" or "adjacent" in the context of
an oligonucleotide refers to nucleosides, nucleobases, sugar
moieties, or internucleoside linkages that are immediately adjacent
to each other independent of the other moieties of the
oligonucleotide. For example, "contiguous nucleobases" means
nucleobases that are immediately adjacent to each other in a
sequence. Moieties that are "directly linked" are immediately
adjacent to each other and not separated by any other type of
moiety.
[0029] As used herein, "degradation" in the context of a nucleic
acid or protein means at least one cleavage of a contiguous nucleic
acid or polypeptide. In certain embodiments, the at least one
cleavage is performed by a nuclease.
[0030] As used herein, "double-stranded antisense compound" means
an antisense compound comprising two oligomeric compounds that are
complementary to each other and form a duplex, and wherein one of
the two said oligomeric compounds comprises an antisense
oligonucleotide.
[0031] As used herein, "effective amount" means the amount of
compound sufficient to effectuate a desired physiological outcome
in a subject in need of the compound. The effective amount may vary
among subjects depending on the health and physical condition of
the subject to be treated, the taxonomic group of the subjects to
be treated, the formulation of the composition, assessment of the
subject's medical condition, and other relevant factors.
[0032] As used herein, "efficacy" means the ability to produce a
desired effect.
[0033] As used herein, "exon-exon junction" means a contiguous
portion of an mRNA where two exons of a corresponding pre-mRNA were
spliced together. An exon-exon junction includes at least one
nucleoside of each of the two respective exons and may include up
to the entirety of both of the respective exons.
[0034] As used herein, "expression" includes all the functions by
which a gene's coded information is converted into structures
present and operating in a cell. Such structures include, but are
not limited to, the products of transcription and translation. As
used herein, "modulation of expression" means any change in amount
or activity of a product of transcription or translation of a gene.
Such a change may be an increase or a reduction of any amount
relative to the expression level prior to the modulation.
[0035] As used herein, "gapmer" means an oligonucleotide or a
portion of an oligonucleotide having a central region comprising a
plurality of nucleosides that support RNase H cleavage positioned
between a 5'-region and a 3'-region. Herein, the 3'- and 5'-most
nucleosides of the central region each comprise a 2'-deoxyfuranosyl
sugar moiety. Herein, the 3'-most nucleoside of the 5'-region
comprises a 2'-modified sugar moiety or a sugar surrogate. Herein,
the 5'-most nucleoside of the 3'-region comprises a 2'-modified
sugar moiety or a sugar surrogate. The "central region" may be
referred to as a "gap"; and the "5'-region" and "3'-region" may be
referred to as "wings".
[0036] As used herein, "hybridization" means the pairing or
annealing of complementary oligonucleotides and/or nucleic acids.
While not limited to a particular mechanism, the most common
mechanism of hybridization involves hydrogen bonding, which may be
Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,
between complementary nucleobases.
[0037] As used herein, "inhibiting the expression or activity"
refers to a reduction or blockade of the expression or activity
relative to the expression or activity in an untreated or control
sample and does not necessarily indicate a total elimination of
expression or activity. Inhibition of the expression or activity of
a nucleic acid, such as a target mRNA, includes but is not limited
to degradation of the nucleic acid.
[0038] As used herein, the terms "internucleoside linkage" means a
group or bond that forms a covalent linkage between adjacent
nucleosides in an oligonucleotide. As used herein "modified
internucleoside linkage" means any internucleoside linkage other
than a naturally occurring, phosphodiester internucleoside linkage.
"Phosphorothioate linkage" means a modified internucleoside linkage
in which one of the non-bridging oxygen atoms of a phosphodiester
is replaced with a sulfur atom. Modified internucleoside linkages
may or may not contain a phosphorus atom. A "neutral
internucleoside linkage" is a modified internucleoside linkage that
is mostly or completely uncharged at pH 7.4 and/or has a pKa below
7.4.
[0039] As used herein, "abasic nucleoside" means a sugar moiety in
an oligonucleotide or oligomeric compound that is not directly
connected to a nucleobase. In certain embodiments, an abasic
nucleoside is adjacent to one or two nucleosides in an
oligonucleotide.
[0040] As used herein, "LICA-1" is a conjugate group that is
represented by the formula:
##STR00001##
[0041] As used herein, "linker-nucleoside" means a nucleoside that
links, either directly or indirectly, an oligonucleotide to a
conjugate moiety. Linker-nucleosides are located within the
conjugate linker of an oligomeric compound. Linker-nucleosides are
not considered part of the oligonucleotide portion of an oligomeric
compound even if they are contiguous with the oligonucleotide.
[0042] As used herein, "non-bicyclic sugar" or "non-bicyclic sugar
moiety" means a sugar moiety that comprises fewer than 2 rings.
Substituents of modified, non-bicyclic sugar moieties do not form a
bridge between two atoms of the sugar moiety to form a second
ring.
[0043] As used herein, "linked nucleosides" are nucleosides that
are connected in a continuous sequence (i.e. no additional
nucleosides are present between those that are linked).
[0044] As used herein, "mismatch" or "non-complementary" means a
nucleobase of a first oligonucleotide that is not complementary
with the corresponding nucleobase of a second oligonucleotide or
target nucleic acid when the first and second oligomeric compound
are aligned.
[0045] As used herein, "modulating" refers to changing or adjusting
a feature in a cell, tissue, organ or organism.
[0046] As used herein, "MOE" means methoxyethyl. "2'-MOE" or
"2'-O-methoxyethyl" means a 2'-OCH.sub.2CH.sub.2OCH.sub.3 group at
the 2'-position of a furanosyl ring. In certain embodiments, the
2'-OCH.sub.2CH.sub.2OCH.sub.3 group is in place of the 2'-OH group
of a ribosyl ring or in place of a 2'-H in a 2'-deoxyribosyl
ring.
[0047] As used herein, "motif" means the pattern of unmodified
and/or modified sugar moieties, nucleobases, and/or internucleoside
linkages, in an oligonucleotide or a portion of an
oligonucleotide.
[0048] As used herein, "naturally occurring" means found in
nature.
[0049] As used herein, "nucleobase" means an unmodified nucleobase
or a modified nucleobase. As used herein an "unmodified nucleobase"
is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine
(G). As used herein, a modified nucleobase is a group of atoms
capable of pairing with at least one unmodified nucleobase. A
universal base is a nucleobase that can pair with any one of the
five unmodified nucleobases. 5-methylcytosine (.sup.mC) is one
example of a modified nucleobase.
[0050] As used herein, "nucleobase sequence" means the order of
contiguous nucleobases in a nucleic acid or oligonucleotide
independent of any sugar moiety or internucleoside linkage
modification.
[0051] As used herein, "nucleoside" means a moiety comprising a
nucleobase and a sugar moiety. The nucleobase and sugar moiety are
each, independently, unmodified or modified. As used herein,
"modified nucleoside" means a nucleoside comprising a modified
nucleobase and/or a modified sugar moiety.
[0052] As used herein, "oligomeric compound" means a compound
consisting of an oligonucleotide and optionally one or more
additional features, such as a conjugate group or terminal
group.
[0053] As used herein, "oligonucleotide" means a strand of linked
nucleosides connected via internucleoside linkages, wherein each
nucleoside and internucleoside linkage may be modified or
unmodified. Unless otherwise indicated, oligonucleotides consist of
8-50 linked nucleosides. As used herein, "modified oligonucleotide"
means an oligonucleotide, wherein at least one nucleoside or
internucleoside linkage is modified. As used herein, "unmodified
oligonucleotide" means an oligonucleotide that does not comprise
any nucleoside modifications or internucleoside modifications.
[0054] As used herein, "pharmaceutically acceptable carrier or
diluent" means any substance suitable for use in administering to
an animal. Certain such carriers enable pharmaceutical compositions
to be formulated as, for example, liquids, powders, or suspensions
that can be aerosolized or otherwise dispersed for inhalation by a
subject. In certain embodiments, a pharmaceutically acceptable
carrier or diluent is sterile water; sterile saline; or sterile
buffer solution.
[0055] As used herein "pharmaceutically acceptable salts" means
physiologically and pharmaceutically acceptable salts of compounds,
such as oligomeric compounds, i.e., salts that retain the desired
biological activity of the compound and do not impart undesired
toxicological effects thereto.
[0056] As used herein "pharmaceutical composition" means a mixture
of substances suitable for administering to a subject. For example,
a pharmaceutical composition may comprise an antisense compound and
an aqueous solution.
[0057] As used herein, "RNAi compound" means an antisense compound
that acts, at least in part, through RISC or Ago2 to modulate a
target nucleic acid and/or protein encoded by a target nucleic
acid. RNAi compounds include, but are not limited to
double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA,
including microRNA mimics. In certain embodiments, an RNAi compound
modulates the amount, activity, and/or splicing of a target nucleic
acid. The term RNAi compound excludes antisense oligonucleotides
that act through RNase H.
[0058] As used herein, the term "single-stranded" in reference to
an antisense compound means such a compound consisting of one
oligomeric compound that is not paired with a second oligomeric
compound to form a duplex. "Self-complementary" in reference to an
oligonucleotide means an oligonucleotide that at least partially
hybridizes to itself. A compound consisting of one oligomeric
compound, wherein the oligonucleotide of the oligomeric compound is
self-complementary, is a single-stranded compound. A
single-stranded antisense or oligomeric compound may be capable of
binding to a complementary oligomeric compound to form a duplex, in
which case the compound would no longer be single-stranded.
[0059] As used herein, "standard cell assay" means an assay
described in any of the Examples, and reasonable variations
thereof.
[0060] As used herein, "stereorandom chiral center" in the context
of a population of molecules of identical molecular formula means a
chiral center having a random stereochemical configuration. For
example, in a population of molecules comprising a stereorandom
chiral center, the number of molecules having the (S) configuration
of the stereorandom chiral center may be but is not necessarily the
same as the number of molecules having the (R) configuration of the
stereorandom chiral center. The stereochemical configuration of a
chiral center is considered random when it is the result of a
synthetic method that is not designed to control the stereochemical
configuration. In certain embodiments, a stereorandom chiral center
is a stereorandom phosphorothioate internucleoside linkage.
[0061] As used herein, "subject" means a human or non-human animal
selected for treatment or therapy.
[0062] As used herein, "sugar moiety" means an unmodified sugar
moiety or a modified sugar moiety. As used herein, "unmodified
sugar moiety" means a .beta.-D-ribosyl moiety, as found in
naturally occurring RNA, or a .beta.-D-2'-deoxyribosyl sugar moiety
as found in naturally occurring DNA. As used herein, "modified
sugar moiety" or "modified sugar" means a sugar surrogate or a
furanosyl sugar moiety other than a .beta.-D-ribosyl or a
.beta.-D-2'-deoxyribosyl. Modified furanosyl sugar moieties may be
modified or substituted at a certain position(s) of the sugar
moiety, or unsubstituted, and they may or may not have a
stereoconfiguration other than .beta.-D-ribosyl. Modified furanosyl
sugar moieties include bicyclic sugars and non-bicyclic sugars. As
used herein, "sugar surrogate" means a modified sugar moiety that
does not comprise a furanosyl or tetrahydrofuranyl ring (is not a
"furanosyl sugar moiety") and that can link a nucleobase to another
group, such as an internucleoside linkage, conjugate group, or
terminal group in an oligonucleotide. Modified nucleosides
comprising sugar surrogates can be incorporated into one or more
positions within an oligonucleotide and such oligonucleotides are
capable of hybridizing to complementary oligomeric compounds or
nucleic acids.
[0063] As used herein, "target" in the context of a nucleic acid,
such as an RNA, means a nucleic acid that an oligomeric compound is
designed to affect. In certain embodiments, an oligomeric compound
comprises an oligonucleotide having a nucleobase sequence that is
complementary to more than one RNA, only one of which is the target
RNA of the oligomeric compound. In certain embodiments, the target
RNA is an RNA present in the species to which an oligomeric
compound is administered. In certain embodiments, the target RNA is
an mRNA. In certain such embodiments, the target mRNA is a mature
mRNA, meaning that the mRNA has already been processed. A mature
mRNA excludes a pre-mRNA.
[0064] As used herein, "therapeutically effective amount" means an
amount of a compound, pharmaceutical agent, or composition that
provides a therapeutic benefit to a subject.
[0065] As used herein, "treat" refers to administering a compound
or pharmaceutical composition to an animal in order to effect an
alteration or improvement of a disease, disorder, or condition in
the animal.
[0066] As used herein, a "standard RNase H cleavage assay" is an
assay wherein a heteroduplex of the modified oligonucleotide and a
complementary strand of unmodified RNA are incubated with each
other to form a heteroduplex, and are then incubated with RNase H1
for specified time points before being analyzed on a polyacrylamide
gel.
[0067] As used herein, a modified nucleoside "supports RNase H
cleavage" when incorporated into an oligonucleotide if RNase H
cleavage of the complementary RNA is observed within two
nucleobases of the modified nucleoside in a standard RNase H
cleavage assay.
Certain Compounds
[0068] In certain embodiments, compounds described herein are
oligomeric compounds comprising or consisting of oligonucleotides
consisting of linked nucleosides. Oligonucleotides may be
unmodified oligonucleotides or may be modified oligonucleotides.
Modified oligonucleotides comprise at least one modification
relative to an unmodified oligonucleotide (i.e., comprise at least
one modified nucleoside (comprising a modified sugar moiety and/or
a modified nucleobase) and/or at least one modified internucleoside
linkage).
Certain Embodiments
[0069] The present disclosure provides the following non-limiting
numbered embodiments: [0070] Embodiment 1. A chirally enriched
population of oligomeric compounds comprising modified
oligonucleotides, wherein the modified oligonucleotides consist of
12-23 linked nucleosides, wherein the modified oligonucleotide
comprises a gapmer consisting of a 5'-region, a central region, and
a 3'-region wherein: the 5'-region consists of 1-5 linked modified
nucleosides, wherein each nucleoside of the 5'-region comprises a
2'-modified furanosyl sugar moiety; the 3'-region consists of 1-5
linked modified nucleosides, wherein each nucleoside of the
3'-region comprises a 2'-modified furanosyl sugar moiety; the
central region consists of 7-10 linked nucleosides, where each
nucleoside of the central region comprises a
2'-.beta.-D-deoxyribosyl sugar moiety; and wherein the central
region has at least one phosphorothioate internucleoside linkage
that is chirally enriched in the (Sp) configuration or the (Rp)
configuration. [0071] Embodiment 2. A chirally enriched population
of oligomeric compounds, wherein the oligomeric compounds comprise
a modified oligonucleotide consisting of 12-23 linked nucleosides,
wherein the modified oligonucleotide is a gapmer consisting of a
5'-region, a central region, and a 3'-region wherein: the 5'-region
consists of 1-5 linked modified nucleosides, wherein each
nucleoside of the 5'-region comprises a 2'-modified furanosyl sugar
moiety; the 3'-region consists of 1-5 linked modified nucleosides,
wherein each nucleoside of the 3'-region comprises a 2'-modified
furanosyl sugar moiety; the central region consists of 7-10 linked
nucleosides, where each nucleoside of the central region comprises
a 2'-.beta.-D-deoxyribosyl sugar moiety; wherein the central region
has at least one phosphorothioate internucleoside linkage; and
wherein a percentage of oligomeric compounds within the population
that contain a stereochemical configuration at the phosphorothioate
internucleoside linkage is greater than an expected percentage of
oligomeric compounds expected to contain the same particular
stereochemical configuration at the phosphorothioate
internucleoside linkage within the population if the stereochemical
configuration at the phosphorothioate internucleoside linkage was
stereorandom. [0072] Embodiment 3. The chirally enriched population
of oligomeric compounds of embodiment 1 or 2, wherein each central
region internucleoside linkage is selected from among a
phosphodiester internucleoside linkage and a phosphorothioate
internucleoside linkage. [0073] Embodiment 4. The chirally enriched
population of oligomeric compounds of embodiment 1 or 2, wherein
the central region has 2 phosphorothioate internucleoside linkages.
[0074] Embodiment 5. The chirally enriched population of oligomeric
compounds of embodiment 1 or 2, wherein the central region has 3
phosphorothioate internucleoside linkages. [0075] Embodiment 6. The
chirally enriched population of oligomeric compounds of embodiment
1 or 2, wherein the central region has 4 phosphorothioate
internucleoside linkages. [0076] Embodiment 7. The chirally
enriched population of oligomeric compounds of embodiment 1 or 2,
wherein the central region has 5 phosphorothioate internucleoside
linkages. [0077] Embodiment 8. The chirally enriched population of
oligomeric compounds of embodiment 1 or 2, wherein the central
region has 6 phosphorothioate internucleoside linkages. [0078]
Embodiment 9. The chirally enriched population of oligomeric
compounds of embodiment 1 or 2, wherein the central region has 7
phosphorothioate internucleoside linkages. [0079] Embodiment 10.
The chirally enriched population of oligomeric compounds of
embodiment 1 or 2, wherein the central region has 8
phosphorothioate internucleoside linkages.
[0080] Embodiment 11. The chirally enriched population of
oligomeric compounds of embodiment 1 or 2, wherein the central
region has 9 phosphorothioate internucleoside linkages. [0081]
Embodiment 12. The chirally enriched population of oligomeric
compounds of embodiment 1 or 2, wherein the central region has 10
phosphorothioate internucleoside linkages. [0082] Embodiment 13.
The chirally enriched population of oligomeric compounds of
embodiment 1 or 2, wherein the central region has 11
phosphorothioate internucleoside linkages. [0083] Embodiment 14.
The chirally enriched population of oligomeric compounds of
embodiment 1 or 2, wherein each central region internucleoside
linkage is a phosphorothioate internucleoside linkage. [0084]
Embodiment 15. The chirally enriched population of oligomeric
compounds of embodiment 1 or 2, wherein each phosphorothioate
internucleoside linkage has the (Sp) configuration. [0085]
Embodiment 16. The chirally enriched population of oligomeric
compounds of embodiment 1 or 2, wherein each phosphorothioate
internucleoside linkage has the (Rp) configuration. [0086]
Embodiment 17. The chirally enriched population of oligomeric
compounds of embodiment 1 or 2, wherein 1 central region
internucleoside linkage has the (Rp) configuration. [0087]
Embodiment 18. The chirally enriched population of oligomeric
compounds of any of embodiments 1-14, wherein 2 central region
internucleoside linkages have the (Rp) configuration. [0088]
Embodiment 19. The chirally enriched population of oligomeric
compounds of embodiment 18, wherein the 2 central region
internucleoside linkages having the (Rp) configuration are
adjacent. [0089] Embodiment 20. The chirally enriched population of
oligomeric compounds of embodiment 1 or 2, wherein the central
region consists of 7 linked nucleosides and has the formula: [0090]
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7; wherein each N.sub.d is a
2'-.beta.-D- deoxyribosyl sugar moiety and each of L1, L2, L3, L4,
L5, L6, and L7 is a phosphorothioate internucleoside linkage; and
wherein at least one of L1, L2, L3, L4, L5, L6, and L7 has the (Sp)
configuration or the (Rp) configuration. [0091] Embodiment 21. The
chirally enriched population of oligomeric compounds of embodiment
20, wherein L1 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L2, L3, L4, L5, L6, and L7
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0092] Embodiment 22. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein L2 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L3, L4, L5, L6, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0093] Embodiment 23. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein L3 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L4, L5, L6, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0094] Embodiment 24. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein L4 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L5, L6, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0095] Embodiment 25. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein L5 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L6, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0096] Embodiment 26. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein L6 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0097] Embodiment 27. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein L7 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, and L6 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0098] Embodiment 28. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein each
L1 and L2 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L3, L4, L5, L6, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0099] Embodiment 29. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein each
L2 and L3 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L4, L5, L6, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0100] Embodiment 30. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein each
L3 and L4 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L5, L6, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0101] Embodiment 31. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein each
L4 and L5 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L3, L6, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0102] Embodiment 32. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein each
L5 and L6 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L3, L4, and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0103] Embodiment 33. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein each
L6 and L7 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L3, L4, and L5 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0104] Embodiment 34. The chirally enriched
population of oligomeric compounds of embodiment 20, wherein each
L1 is a phosphorothioate internucleoside linkage having the (Rp)
configuration, each L2 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L3, L4,
L5, L6 and L7 is a stereorandom phosphorothioate internucleoside
linkage. [0105] Embodiment 35. The chirally enriched population of
oligomeric compounds of embodiment 20, wherein each L2 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L3 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L4,
L5, L6 and L7 is a stereorandom phosphorothioate internucleoside
linkage. [0106] Embodiment 36. The chirally enriched population of
oligomeric compounds of embodiment 20, wherein each L3 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L4 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L2,
L5, L6 and L7 is a stereorandom phosphorothioate internucleoside
linkage. [0107] Embodiment 37. The chirally enriched population of
oligomeric compounds of embodiment 20, wherein each L4 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L5 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L2,
L3, L6 and L7 is a stereorandom phosphorothioate internucleoside
linkage. [0108] Embodiment 38. The chirally enriched population of
oligomeric compounds of embodiment 20, wherein each L5 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L6 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L2,
L3, L4 and L7 is a stereorandom phosphorothioate internucleoside
linkage. [0109] Embodiment 39. The chirally enriched population of
oligomeric compounds of embodiment 20, wherein each L6 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L7 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L2,
L3, L4 and L5 is a stereorandom phosphorothioate internucleoside
linkage. [0110] Embodiment 40. The oligomeric compound of
embodiment 1 or 2, wherein the central region consists of 8 linked
nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7(N.sub.d).sub.L8; wherein
each N.sub.d is a 2'-.beta.-D-deoxyribosyl sugar moiety and each of
L1, L2, L3, L4, L5, L6, L7, and L8 is a phosphorothioate
internucleoside linkage; and wherein at least one of L1, L2, L3,
L4, L5, L6, L7 and L8 has the (Sp) configuration or the (Rp)
configuration. [0111] Embodiment 41. The oligomeric compound of
embodiment 1 or 2, wherein the central region consists of 9 linked
nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7(N.sub.d).sub.L8(N.sub.d).sub.L9;
wherein each N.sub.d is a 2'-.beta.-D-deoxyribosyl sugar moiety and
each of L1, L2, L3, L4, L5, L6, L7, L8, and L9 is a
phosphorothioate internucleoside linkage; and wherein at least one
of L1, L2, L3, L4, L5, L6, L7, L8 and L9 has the (Sp) configuration
or the (Rp) configuration. [0112] Embodiment 42. The chirally
enriched population of oligomeric compounds of embodiment 41,
wherein L1 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L2, L3, L4, L5, L6, L7 and L8
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0113] Embodiment 43. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein L2 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L3, L4, L5, L6, L7 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0114] Embodiment 44. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein L3 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L4, L5, L6, L7 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0115] Embodiment 45. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein L4 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L4, L5, L6, L7 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0116] Embodiment 46. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein L5 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L6, L7 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0117] Embodiment 47. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein L6 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L7 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0118] Embodiment 48. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein L7 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0119] Embodiment 49. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein L8 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6 and L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0120] Embodiment 50. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein each
L1 and L2 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L3, L4, L5, L6 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0121] Embodiment 51. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein each
L2 and L3 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L4, L5, L6 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0122] Embodiment 52. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein each
L3 and L4 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L5, L6 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0123] Embodiment 53. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein each
L4 and L5 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L3, L6 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0124] Embodiment 54. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein each
L5 and L6 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L3, L6 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0125] Embodiment 55. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein each
L6 and L7 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L3, L5 and L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0126] Embodiment 56. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein each
L7 and L8 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L1, L2, L3, L5 and L6 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0127] Embodiment 57. The chirally enriched
population of oligomeric compounds of embodiment 41, wherein each
L1 is a phosphorothioate internucleoside linkage having the (Rp)
configuration, each L2 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L3, L4,
L5, L6, L7 and L8 is a stereorandom phosphorothioate
internucleoside linkage. [0128] Embodiment 58. The chirally
enriched population of oligomeric compounds of embodiment 41,
wherein each L2 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L3 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L4, L5, L6, L7 and L8 is a stereorandom
phosphorothioate internucleoside linkage.
[0129] Embodiment 59. The chirally enriched population of
oligomeric compounds of embodiment 41, wherein each L3 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L4 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L2,
L5, L6, L7 and L8 is a stereorandom phosphorothioate
internucleoside linkage. [0130] Embodiment 60. The chirally
enriched population of oligomeric compounds of embodiment 41,
wherein each L4 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L5 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L6, L7 and L8 is a stereorandom
phosphorothioate internucleoside linkage. [0131] Embodiment 61. The
chirally enriched population of oligomeric compounds of embodiment
41, wherein each L5 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L6 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L4, L7 and L8 is a stereorandom
phosphorothioate internucleoside linkage. [0132] Embodiment 62. The
chirally enriched population of oligomeric compounds of embodiment
41, wherein each L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L4, L5 and L8 is a stereorandom
phosphorothioate internucleoside linkage. [0133] Embodiment 63. The
chirally enriched population of oligomeric compounds of embodiment
41, wherein each L7 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L8 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L4, L5 and L6 is a stereorandom
phosphorothioate internucleoside linkage. [0134] Embodiment 64. The
oligomeric compound of embodiment 1 or 2, wherein the central
region consists of 9 linked nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7(N.sub.d).sub.L8(N.sub.d).sub.L9;
wherein each N.sub.d is a 2'-.beta.-D-deoxyribosyl sugar moiety and
each of L1, L2, L3, L4, L5, L6, L7, L8, and L9 is a
phosphorothioate internucleoside linkage; and wherein at least one
of L1, L2, L3, L4, L5, L6, L7, L8 and L9 has the (Sp) configuration
or the (Rp) configuration. [0135] Embodiment 65. The chirally
enriched population of oligomeric compounds of embodiment 51 or 64,
wherein L1 is a phosphorothioate internucleoside linkage having the
(Rp) configuration and where each of L2, L3, L4, L5, L6, L7, L8 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0136] Embodiment 66. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein L2 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L3, L4, L5, L6, L7, L8 and L9
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0137] Embodiment 67. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein L3 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L4, L5, L6, L7, L8 and L9
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0138] Embodiment 68. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein L4 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L5, L6, L7, L8 and L9
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0139] Embodiment 69. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein L5 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L6, L7, L8 and L9
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0140] Embodiment 70. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein L6 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L7, L8 and L9
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0141] Embodiment 71. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein L7 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6, L8 and L9
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0142] Embodiment 72. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein L8 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6, L7 and L9
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0143] Embodiment 73. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein L9 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6, L7 and L8
is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0144] Embodiment 74. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
of L1 and L2 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L3, L4, L5, L6, L7, L8 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0145] Embodiment 75. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
of L2 and L3 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L4, L5, L6, L7, L8 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0146] Embodiment 76. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
of L3 and L4 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L5, L6, L7, L8 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0147] Embodiment 77. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
of L4 and L5 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L6, L7, L8 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0148] Embodiment 78. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
of L5 and L6 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L7, L8 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0149] Embodiment 79. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
of L6 and L7 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L5, L8 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0150] Embodiment 80. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
of L7 and L8 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L5, L6 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0151] Embodiment 81. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
of L8 and L9 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L5, L6 and
L7 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0152] Embodiment 82. The chirally enriched
population of oligomeric compounds of embodiment 64, wherein each
L1 is a phosphorothioate internucleoside linkage having the (Rp)
configuration, each L2 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L3, L4,
L5, L6, L7, L8 and L9 is a stereorandom phosphorothioate
internucleoside linkage. [0153] Embodiment 83. The chirally
enriched population of oligomeric compounds of embodiment 64,
wherein each L2 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L3 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L4, L5, L6, L7, L8 and L9 is a stereorandom
phosphorothioate internucleoside linkage. [0154] Embodiment 84. The
chirally enriched population of oligomeric compounds of embodiment
64, wherein each
[0155] L3 is a phosphorothioate internucleoside linkage having the
(Rp) configuration, each L4 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L2,
L5, L6, L7, L8 and L9 is a stereorandom phosphorothioate
internucleoside linkage. [0156] Embodiment 85. The chirally
enriched population of oligomeric compounds of embodiment 64,
wherein each L4 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L5 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L6, L7, L8 and L9 is a stereorandom
phosphorothioate internucleoside linkage. [0157] Embodiment 86. The
chirally enriched population of oligomeric compounds of embodiment
64, wherein each L5 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L6 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L4, L7, L8 and L9 is a stereorandom
phosphorothioate internucleoside linkage. [0158] Embodiment 87. The
chirally enriched population of oligomeric compounds of embodiment
64, wherein each L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L4, L5, L8 and L9 is a stereorandom
phosphorothioate internucleoside linkage. [0159] Embodiment 88. The
chirally enriched population of oligomeric compounds of embodiment
64, wherein each L7 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L8 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L4, L5, L6 and L9 is a stereorandom
phosphorothioate internucleoside linkage. [0160] Embodiment 89. The
chirally enriched population of oligomeric compounds of embodiment
64, wherein each
[0161] L8 is a phosphorothioate internucleoside linkage having the
(Rp) configuration, each L9 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L2,
L3, L4, L5, L6 and L7 is a stereorandom phosphorothioate
internucleoside linkage. [0162] Embodiment 90. The oligomeric
compound of embodiment 1 or 2, wherein the central region consists
of 10 linked nucleosides and has the formula:
(N.sub.d).sub.L1(N.sub.d).sub.L2(N.sub.d).sub.L3(N.sub.d).sub.L4(N.sub.d)-
.sub.L5(N.sub.d).sub.L6(N.sub.d).sub.L7(N.sub.d).sub.L8(N.sub.d).sub.L9(N.-
sub.d).sub.L10; wherein each N.sub.d is a 2'-.beta.-D-deoxyribosyl
sugar moiety and each of L1, L2, L3, L4, L5, L6, L7, L8, L9, and
L10 is a phosphorothioate internucleoside linkage; and wherein at
least one of L1, L2, L3, L4, L5, L6, L7, L8, L9 and L10 has the
(Sp) configuration or the (Rp) configuration. [0163] Embodiment 91.
The chirally enriched population of oligomeric compounds of
embodiment 69 or 90, wherein L1 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L2, L3, L4, L5, L6, L7, L8, L9 and L10 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0164] Embodiment 92. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L2 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L3, L4, L5, L6, L7, L8, L9 and
L10 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0165] Embodiment 93. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L3 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L4, L5, L6, L7, L8, L9 and
L10 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0166] Embodiment 94. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L4 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L5, L6, L7, L8, L9 and
L10 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0167] Embodiment 95. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L5 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L6, L7, L8, L9 and
L10 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0168] Embodiment 96. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L6 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L7, L8, L9 and
L10 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0169] Embodiment 97. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L7 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6, L8, L9 and
L10 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0170] Embodiment 98. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L8 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6, L7, L9 and
L10 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0171] Embodiment 99. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L9 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6, L7, L8 and
L10 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0172] Embodiment 100. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein L10 is
a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L2, L3, L4, L5, L6, L7, L8 and
L9 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0173] Embodiment 101. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L1 and L2 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L3, L4, L5, L6, L7, L8, L9
and L10 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0174] Embodiment 102. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L2 and L3 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L4, L5, L6, L7, L8, L9
and L10 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0175] Embodiment 103. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L3 and L4 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L5, L6, L7, L8, L9
and L10 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0176] Embodiment 104. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L4 and L5 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L6, L7, L8, L9
and L10 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0177] Embodiment 105. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L5 and L6 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L7, L8, L9
and L10 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0178] Embodiment 106. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L6 and L7 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L5, L8, L9
and L10 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0179] Embodiment 107. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L7 and L8 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L5, L6, L9
and L10 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0180] Embodiment 108. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L8 and L9 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L5, L6, L7
and L10 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0181] Embodiment 109. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L9 and L10 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L5, L6, L7
and L8 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0182] Embodiment 110. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
of L3, L6, L9 and L10 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L4, L5, L7,
and L8 is a phosphorothioate internucleoside linkage having the
(Sp) configuration. [0183] Embodiment 111. The chirally enriched
population of oligomeric compounds of embodiment 90, wherein each
L1 is a phosphorothioate internucleoside linkage having the (Rp)
configuration, each L2 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L3, L4,
L5, L6, L7, L8, L9 and L10 is a stereorandom phosphorothioate
internucleoside linkage. [0184] Embodiment 112. The chirally
enriched population of oligomeric compounds of embodiment 90,
wherein each L2 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L3 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L4, L5, L6, L7, L8, L9 and L10 is a stereorandom
phosphorothioate internucleoside linkage. [0185] Embodiment 113.
The chirally enriched population of oligomeric compounds of
embodiment 90, wherein each L3 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L4 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L5, L6, L7, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage. [0186]
Embodiment 114. The chirally enriched population of oligomeric
compounds of embodiment 90, wherein each L4 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L5 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L6, L7, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage. [0187]
Embodiment 115. The chirally enriched population of oligomeric
compounds of embodiment 90, wherein each L5 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L6 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L7, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage. [0188]
Embodiment 116. The chirally enriched population of oligomeric
compounds of embodiment 90, wherein each L6 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L7 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L8, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage. [0189]
Embodiment 117. The chirally enriched population of oligomeric
compounds of embodiment 90, wherein each L7 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L9 and L10
is a stereorandom phosphorothioate internucleoside linkage. [0190]
Embodiment 118. The chirally enriched population of oligomeric
compounds of embodiment 90, wherein each L8 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L9 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L7 and L10
is a stereorandom phosphorothioate internucleoside linkage. [0191]
Embodiment 119. The chirally enriched population of oligomeric
compounds of embodiment 90, wherein each L9 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L10 is
a phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L7 and L8
is a stereorandom phosphorothioate internucleoside linkage.
Embodiment 120. The oligomeric compound of embodiment 1 or 2,
wherein the central region consists of 11 linked nucleosides and
has the formula: [0192]
(Nd)L1(Nd)L2(Nd)L3(Nd)L4(Nd)L5(Nd)L6(Nd)L7(Nd)L8(Nd)L9(Nd)L10(Nd)L11;
wherein, each Nd is a 2'-.beta.-D-deoxyribosyl sugar moiety and
each of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage; and wherein at least one
of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10 and L11 has the (Sp)
configuration or the (Rp) configuration. [0193] Embodiment 121. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L1 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L2, L3, L4, L5, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0194] Embodiment 122. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L2 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L3, L4, L5, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0195] Embodiment 123. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L3 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L4, L5, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0196] Embodiment 124. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L4 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L5, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0197] Embodiment 125. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L5 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L6,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0198] Embodiment 126. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0199] Embodiment 127. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L7 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0200] Embodiment 128. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L8 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0201] Embodiment 129. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L9 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L8, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0202] Embodiment 130. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L10 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L8, L9 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0203] Embodiment 131. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein L11 is a phosphorothioate internucleoside linkage
having the (Rp) configuration and where each of L1, L2, L3, L4, L5,
L6, L7, L8, L9 and L10 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0204] Embodiment 132. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein each of L1 and L2 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L3, L4, L5, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration.
[0205] Embodiment 133. The chirally enriched population of
oligomeric compounds of embodiment 120, wherein each of L2 and L3
is a phosphorothioate internucleoside linkage having the (Rp)
configuration and where each of L1, L4, L5, L6, L7, L8, L9, L10 and
L11 is a phosphorothioate internucleoside linkage having the (Sp)
configuration. [0206] Embodiment 134. The chirally enriched
population of oligomeric compounds of embodiment 120, wherein each
of L2 and L3 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L4, L5, L6, L7, L8,
L9, L10 and L11 is a phosphorothioate internucleoside linkage
having the (Sp) configuration. [0207] Embodiment 135. The chirally
enriched population of oligomeric compounds of embodiment 120,
wherein each of L3 and L4 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L2, L5,
L6, L7, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0208] Embodiment 136. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein each of L4 and L5 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L6, L7, L8, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0209] Embodiment 137. The chirally enriched
population of oligomeric compounds of embodiment 120, wherein each
of L5 and L6 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L7, L8,
L9, L10 and L11 is a phosphorothioate internucleoside linkage
having the (Sp) configuration. [0210] Embodiment 138. The chirally
enriched population of oligomeric compounds of embodiment 120,
wherein each of L6 and L7 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L2, L3,
L4, L5, L8, L9, L10 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0211] Embodiment 139. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein each of L7 and L8 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L9, L10 and L11 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration. [0212] Embodiment 140. The chirally enriched
population of oligomeric compounds of embodiment 120, wherein each
of L8 and L9 is a phosphorothioate internucleoside linkage having
the (Rp) configuration and where each of L1, L2, L3, L4, L5, L6,
L7, L10 and L11 is a phosphorothioate internucleoside linkage
having the (Sp) configuration. [0213] Embodiment 141. The chirally
enriched population of oligomeric compounds of embodiment 120,
wherein each of L9 and L10 is a phosphorothioate internucleoside
linkage having the (Rp) configuration and where each of L1, L2, L3,
L4, L5, L6, L7, L9 and L11 is a phosphorothioate internucleoside
linkage having the (Sp) configuration. [0214] Embodiment 142. The
chirally enriched population of oligomeric compounds of embodiment
120, wherein each of L10 and L11 is a phosphorothioate
internucleoside linkage having the (Rp) configuration and where
each of L1, L2, L3, L4, L5, L6, L7, L8 and L9 is a phosphorothioate
internucleoside linkage having the (Sp) configuration. [0215]
Embodiment 143. The chirally enriched population of oligomeric
compounds of embodiment 120, wherein each L1 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L2 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L3, L4, L5, L6, L7, L8, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside linkage.
[0216] Embodiment 144. The chirally enriched population of
oligomeric compounds of embodiment 120, wherein each L2 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L3 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L4,
L5, L6, L7, L8, L9, L10 and L11 is a stereorandom phosphorothioate
internucleoside linkage. [0217] Embodiment 145. The chirally
enriched population of oligomeric compounds of embodiment 120,
wherein each L3 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L4 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L5, L6, L7, L8, L9, L10 and L11 is a stereorandom
phosphorothioate internucleoside linkage. [0218] Embodiment 146.
The chirally enriched population of oligomeric compounds of
embodiment 120, wherein each L4 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L5 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L6, L7, L8, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside linkage.
[0219] Embodiment 147. The chirally enriched population of
oligomeric compounds of embodiment 120, wherein each L5 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L6 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and wherein each of L1, L2,
L3, L4, L7, L8, L9, L10 and L11 is a stereorandom phosphorothioate
internucleoside linkage. [0220] Embodiment 148. The chirally
enriched population of oligomeric compounds of embodiment 120,
wherein each L6 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L7 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L4, L5, L8, L9, L10 and L11 is a stereorandom
phosphorothioate internucleoside linkage. [0221] Embodiment 149.
The chirally enriched population of oligomeric compounds of
embodiment 120, wherein each L7 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L8 is a
phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L9, L10
and L11 is a stereorandom phosphorothioate internucleoside linkage.
[0222] Embodiment 150. The chirally enriched population of
oligomeric compounds of embodiment 120, wherein each L8 is a
phosphorothioate internucleoside linkage having the (Rp)
configuration, each L9 is a phosphorothioate internucleoside
linkage having the (Sp) configuration, and where each of L1, L2,
L3, L4, L5, L6, L7, L10 and L11 is a stereorandom phosphorothioate
internucleoside linkage. [0223] Embodiment 151. The chirally
enriched population of oligomeric compounds of embodiment 120,
wherein each L9 is a phosphorothioate internucleoside linkage
having the (Rp) configuration, each L10 is a phosphorothioate
internucleoside linkage having the (Sp) configuration, and where
each of L1, L2, L3, L4, L5, L6, L7, L8 and L11 is a stereorandom
phosphorothioate internucleoside linkage. [0224] Embodiment 152.
The chirally enriched population of oligomeric compounds of
embodiment 120, wherein each L10 is a phosphorothioate
internucleoside linkage having the (Rp) configuration, each L11 is
a phosphorothioate internucleoside linkage having the (Sp)
configuration, and where each of L1, L2, L3, L4, L5, L6, L7, L8 and
L9 is a stereorandom phosphorothioate internucleoside linkage.
[0225] Embodiment 153. The chirally enriched population of
oligomeric compounds of any of embodiments 1-152, wherein the
5'-region consists of 2-4 linked nucleosides. [0226] Embodiment
154. The chirally enriched population of oligomeric compounds of
any of embodiments 1-152, wherein the 5'-region consists of 1
nucleoside. [0227] Embodiment 155. The chirally enriched population
of oligomeric compounds of any of embodiments 1-152, wherein the
5'-region consists of 2 linked nucleosides. [0228] Embodiment 156.
The chirally enriched population of oligomeric compounds of any of
embodiments 1-152, wherein the 5'-region consists of 3 linked
nucleosides. [0229] Embodiment 157. The chirally enriched
population of oligomeric compounds of any of embodiments 1-152,
wherein the 5'-region consists of 4 linked nucleosides. [0230]
Embodiment 158. The chirally enriched population of oligomeric
compounds of any of embodiments 1-152, wherein the 5'-region
consists of 5 linked nucleosides. [0231] Embodiment 159. The
chirally enriched population of oligomeric compounds of any of
embodiments 1-158, wherein each nucleoside of the 5'-region
comprises a 2'-modified ribosyl sugar moiety. [0232] Embodiment
160. The chirally enriched population of oligomeric compounds of
any of embodiments 1-159, wherein at least one nucleoside of the
5'-region comprises a bicyclic furanosyl sugar moiety. [0233]
Embodiment 161. The chirally enriched population of oligomeric
compounds of any of embodiments 1-160, wherein each nucleoside of
the 5'-region comprises a bicyclic furanosyl sugar moiety. [0234]
Embodiment 162. The chirally enriched population of oligomeric
compounds of any of embodiments 1-160 wherein at least one
nucleoside of the 5'-region comprises a non-bicyclic sugar moiety.
[0235] Embodiment 163. The chirally enriched population of
oligomeric compounds of embodiment 162, wherein the non-bicyclic
sugar moiety of the at least one nucleoside of the 5'-region is a
2'-substituted ribosyl sugar moiety. [0236] Embodiment 164. The
chirally enriched population of oligomeric compounds of any of
embodiments 1-159 or 162-163, wherein each nucleoside of the
5'-region comprises a non-bicyclic sugar moiety. [0237] Embodiment
165. The chirally enriched population of oligomeric compounds of
embodiment 164, wherein each nucleoside of the 5'-region comprises
a 2'-substituted ribosyl sugar moiety. [0238] Embodiment 166. The
chirally enriched population of oligomeric compounds of any of
embodiments 1-159, wherein each nucleoside of the 5'-region
comprises a 2'-modified furanosyl sugar moiety independently
selected from a bicyclic sugar moiety and a non-bicyclic,
2'-substituted ribosyl sugar moiety. [0239] Embodiment 167. The
chirally enriched population of oligomeric compounds of any of
embodiments 160-163 or 166, wherein each bicyclic sugar moiety of
the 5'-region is selected from among cEt, LNA, and ENA. [0240]
Embodiment 168. The chirally enriched population of oligomeric
compounds of any of embodiments 162-166, wherein each non-bicyclic
sugar moiety of the 5'-region has a 2'-substituent selected from
among 2'-MOE, 2'-OMe, and 2'-NMA. [0241] Embodiment 169. The
chirally enriched population of oligomeric compounds of any of
embodiments 1-168, wherein none of the nucleosides of the 5'-region
comprise a sugar moiety having a 2'-F substituent. [0242]
Embodiment 170. The chirally enriched population of oligomeric
compounds of any of embodiments 1-169, wherein each nucleobase of
the 5'-region is independently selected from among thymine, uracil,
guanine, cytosine, 5-methylcytosine, and adenine. [0243] Embodiment
171. The chirally enriched population of oligomeric compounds of
any of embodiments 1-170, wherein each internucleoside linkage of
the 5'-region is selected from among phosphodiester and
phosphorothioate internucleoside linkages. [0244] Embodiment 172.
The chirally enriched population of oligomeric compounds of any of
embodiments 1-171, wherein the 3'-region consists of 2-4 linked
nucleosides. [0245] Embodiment 173. The chirally enriched
population of oligomeric compounds of any of embodiments 1-171,
wherein the 3'-region consists of 1 nucleoside. [0246] Embodiment
174. The chirally enriched population of oligomeric compounds of
any of embodiments 1-171, wherein the 3'-region consists of 2
linked nucleosides. [0247] Embodiment 175. The chirally enriched
population of oligomeric compounds of any of embodiments 1-171,
wherein the 3'-region consists of 3 linked nucleosides. [0248]
Embodiment 176. The chirally enriched population of oligomeric
compounds of any of embodiments 1-170, wherein the 3'-region
consists of 4 linked nucleosides. [0249] Embodiment 177. The
chirally enriched population of oligomeric compounds of any of
embodiments 1-171, wherein the 3'-region consists of 5 linked
nucleosides. [0250] Embodiment 178. The chirally enriched
population of oligomeric compounds of any of embodiments 1-177,
wherein each nucleoside of the 3'-region comprises a 2'-modified
ribosyl sugar moiety. [0251] Embodiment 179. The chirally enriched
population of oligomeric compounds of any of embodiments 1-178,
wherein at least one nucleoside of the 3'-region comprises a
bicyclic furanosyl sugar moiety. [0252] Embodiment 180. The
chirally enriched population of oligomeric compounds of any of
embodiments 1-179, wherein each nucleoside of the 3'-region
comprises a bicyclic furanosyl sugar moiety. [0253] Embodiment 181.
The chirally enriched population of oligomeric compounds of any of
embodiments 1-179, wherein at least one nucleoside of the 3'-region
comprises a non-bicyclic sugar moiety. [0254] Embodiment 182. The
chirally enriched population of oligomeric compounds of embodiment
1-181, wherein the non-bicyclic sugar moiety of the at least one
nucleoside of the 3'-region is a 2'-substituted ribosyl sugar
moiety. [0255] Embodiment 183. The chirally enriched population of
oligomeric compounds of any of embodiments 1-178 or 181-182,
wherein each nucleoside of the 3'-region comprises a non-bicyclic
sugar moiety. [0256] Embodiment 184. The chirally enriched
population of oligomeric compounds of embodiment 183, wherein each
nucleoside of the 3'-region comprises a 2'-substituted ribosyl
sugar moiety. [0257] Embodiment 185. The chirally enriched
population of oligomeric compounds of any of embodiments 1-178,
wherein each nucleoside of the 3'-region comprises a 2'-modified
furanosyl sugar moiety independently selected from a bicyclic sugar
moiety and a non-bicyclic, 2'-substituted ribosyl sugar moiety.
[0258] Embodiment 186. The chirally enriched population of
oligomeric compounds of any of embodiments 179-182 or 185, wherein
each bicyclic sugar moiety of the 3'-region is selected from among
cEt, LNA, and ENA. [0259] Embodiment 187. The chirally enriched
population of oligomeric compounds of any of embodiments 181-185,
wherein each non-bicyclic sugar moiety of the 3'-region has a
2'-substituent selected from among 2'-MOE, 2'-OMe, and 2'-NMA.
[0260] Embodiment 188. The chirally enriched population of
oligomeric compounds of any of embodiments 1-187, wherein none of
the nucleosides of the 3'-region comprise a sugar moiety having a
2'-F substituent. [0261] Embodiment 189. A pharmaceutical
composition comprising, the chirally enriched population of
oligomeric compounds of any of embodiments 1-187. [0262] Embodiment
190. A method comprising, contacting a cell with the chirally
enriched population of oligomeric compounds of any of embodiments
1-187. [0263] Embodiment 191. A method of modulating the amount or
activity of a target nucleic acid in a cell, comprising contacting
a cell with the chirally enriched population of oligomeric
compounds of any of embodiments 1-187.
I. Modifications
A. Modified Nucleosides
[0264] Modified nucleosides comprise a modified sugar moiety, a
modified nucleobase, or both a modifed sugar moiety and a modified
nucleobase.
[0265] 1. Certain Modified Sugar Moieties
[0266] In certain embodiments, sugar moieties are non-bicyclic,
modified furanosyl sugar moieties. In certain embodiments, modified
sugar moieties are bicyclic or tricyclic furanosyl sugar moieties.
In certain embodiments, modified sugar moieties are sugar
surrogates. Such sugar surrogates may comprise one or more
substitutions corresponding to those of other types of modified
sugar moieties.
[0267] In certain embodiments, modified sugar moieties are
non-bicyclic modified furanosyl sugar moieties comprising one or
more acyclic substituent, including but not limited to substituents
at the 2', 3', 4', and/or 5' positions. In certain embodiments, the
furanosyl sugar moiety is a ribosyl sugar moiety. In certain
embodiments one or more acyclic substituent of non-bicyclic
modified sugar moieties is branched. Examples of 2'-substituent
groups suitable for non-bicyclic modified sugar moieties include
but are not limited to: 2'-F, 2'-OCH.sub.3("OMe" or "O-methyl"),
and 2'-O(CH.sub.2).sub.2OCH.sub.3 ("MOE"). In certain embodiments,
2'-substituent groups are selected from among: halo, allyl, amino,
azido, SH, CN, OCN, CF.sub.3, OCF.sub.3, O-C.sub.1-C.sub.10 alkoxy,
O--C.sub.1-C.sub.10 substituted alkoxy, O-C.sub.1-C.sub.10 alkyl,
O-C.sub.1-C.sub.10 substituted alkyl, S-alkyl, N(R.sub.m)-alkyl,
O-alkenyl, S-alkenyl, N(R.sub.m)-alkenyl, O-alkynyl, S-alkynyl,
N(R.sub.m)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl,
O-alkaryl, O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2ON(R.sub.m)(R.sub.n) or
OCH.sub.2C(.dbd.O)-N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H, an amino protecting group, or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl, and the
2'-substituent groups described in Cook et al., U.S. Pat. No.
6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al.,
U.S. Pat. No. 6,005,087. Certain embodiments of these
2'-substituent groups can be further substituted with one or more
substituent groups independently selected from among: hydroxyl,
amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol,
thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.
Examples of 4'-substituent groups suitable for non-bicyclic
modified sugar moieties include but are not limited to alkoxy
(e.g., methoxy), alkyl, and those described in Manoharan et al., WO
2015/106128. Examples of 5'-substituent groups suitable for
non-bicyclic modified sugar moieties include but are not limited
to: 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain
embodiments, non-bicyclic modified sugars comprise more than one
non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar
moieties and the modified sugar moieties and modified nucleosides
described in Migawa et al., WO 2008/101157 and Rajeev et al.,
US2013/0203836.
[0268] In certain embodiments, a 2'-substituted nucleoside or
non-bicyclic 2'-modified nucleoside comprises a sugar moiety
comprising a non-bridging 2'-substituent group selected from: F,
NH.sub.2, N.sub.3, OCF.sub.3, OCH.sub.3, O(CH.sub.2).sub.3NH.sub.2,
CH.sub.2CH.dbd.CH.sub.2, OCH.sub.2CH.dbd.CH.sub.2,
OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2ON(R.sub.m)(R.sub.n),
O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
N-substituted acetamide (OCH.sub.2C(.dbd.O)-N(R.sub.m)(R.sub.n)),
where each R.sub.m and R.sub.n is, independently, H, an amino
protecting group, or substituted or unsubstituted C.sub.1-C.sub.10
alkyl.
[0269] In certain embodiments, a 2'-substituted nucleoside or
non-bicyclic 2'-modified nucleoside comprises a sugar moiety
comprising a non-bridging 2'-substituent group selected from: F,
OCF.sub.3, OCH.sub.3, OCH.sub.2CH.sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ON(CH.sub.3).sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
OCH.sub.2C(.dbd.O)-N(H)CH.sub.3 ("NMA").
[0270] In certain embodiments, a 2'-substituted nucleoside or
non-bicyclic 2'-modified nucleoside comprises a sugar moiety
comprising a non-bridging 2'-substituent group selected from: F,
OCH.sub.3, and OCH.sub.2CH.sub.2OCH.sub.3.
[0271] Certain modifed sugar moieties comprise a bridging sugar
substituent that forms a second ring resulting in a bicyclic sugar
moiety. In certain such embodiments, the bicyclic sugar moiety
comprises a bridge between the 4' and the 2' furanose ring atoms.
In certain such embodiments, the furanose ring is a ribose ring.
Examples of sugar moieties comprising such 4' to 2' bridging sugar
substituents include but are not limited to bicyclic sugars
comprising: 4'-CH.sub.2-2', 4'-(CH.sub.2).sub.2-2',
4'-(CH.sub.2).sub.3-2', 4'-CH.sub.2-O-2' ("LNA"),
4'-CH.sub.2--S-2', 4'-(CH.sub.2).sub.2-O-2' ("ENA"),
4'-CH(CH.sub.3)-O-2' (referred to as "constrained ethyl" or "cEt"
when in the S configuration), 4'-CH.sub.2-O--CH.sub.2-2',
4'-CH.sub.2-N(R)-2', 4'-CH(CH.sub.2OCH.sub.3)-O-2' ("constrained
MOE" or "cMOE") and analogs thereof (see, e.g., Seth et al., U.S.
Pat. No. 7,399,845, Bhat et al., U.S. Pat. No. 7,569,686, Swayze et
al., U.S. Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No.
8,022,193), 4'-C(CH.sub.3)(CH.sub.3)-O-2' and analogs thereof (see,
e.g., Seth et al., U.S. Pat. No. 8,278,283), 4'-CH.sub.2-N(OCH3)-2'
and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No.
8,278,425), 4'-CH.sub.2-O--N(CH.sub.3)-2' (see, e.g., Allerson et
al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No.
8,124,745), 4'-CH.sub.2-C(H)(CH.sub.3)-2' (see, e.g., Zhou, et al.,
J. Org. Chem.,2009, 74, 118-134), 4'-CH.sub.2-C(.dbd.CH.sub.2)-2'
and analogs thereof (see e.g., Seth et al., U.S. Pat. No.
8,278,426), 4'-C(R.sub.aR.sub.b)-N(R)-O-2',
4'-C(R.sub.aR.sub.b)-O--N(R)-2', 4'-CH.sub.2-O--N(R)-2', and
4'-CH.sub.2--N(R)-O-2', wherein each R, R.sub.a, and R.sub.b is,
independently, H, a protecting group, or C.sub.1-C.sub.12 alkyl
(see, e.g. Imanishi et al., U.S. Pat. No. 7,427,672).
[0272] In certain embodiments, such 4' to 2' bridges independently
comprise from 1 to 4 linked groups independently selected from:
--[C(R.sub.a)(R.sub.b)].sub.n--,
--[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.a).dbd.C(R.sub.b)--,
--C(R.sub.a).dbd.N--, --C(.dbd.NR.sub.a)--, --C(.dbd.O)--,
--C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--,
and --N(R.sub.a)--;
[0273] wherein:
[0274] x is 0, 1, or 2;
[0275] n is 1, 2, 3, or 4;
[0276] each R.sub.a and R.sub.b, is, independently, H, a protecting
group, hydroxyl, C.sub.1-C12 alkyl, substituted C.sub.1-C.sub.12
alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12
alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12
alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl,
heterocycle radical, substituted heterocycle radical, heteroaryl,
substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical,
substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl
(C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0277] each J.sub.1 and J.sub.2 is, independently, H,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl
(C(.dbd.O)--H), substituted acyl, a heterocycle radical, a
substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl,
substituted C.sub.1-C.sub.12 aminoalkyl, or a protecting group.
[0278] Additional bicyclic sugar moieties are known in the art,
see, for example: Freier et al., Nucleic Acids Research, 1997,
25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71,
7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin
et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg.
Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem.,
1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 20017,
129, 8362-8379; Elayadi et al.,; Wengel et a., U.S. 7,053,207;
Imanishi et al., U.S. Pat. No. 6,268,490; Imanishi et al. U.S. Pat.
No. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S.
Pat. No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengel
et al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No.
8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al.,
U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582;
and Ramasamy et al., U.S. Pat. No. 6,525,191; Torsten et al., WO
2004/106356;Wengel et al., WO 1999/014226; Seth et al., WO
2007/134181; Seth et al., U.S. Pat. No. 7,547,684; Seth et al.,
U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth
et al., U.S. Pat. No. 7,750,131; Seth et al., U.S. Pat. No.
8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S.
Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et
al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805;
and U.S. Patent Publication Nos. Allerson et al., US2008/0039618
and Migawa et al., US2015/0191727.
[0279] In certain embodiments, bicyclic sugar moieties and
nucleosides incorporating such bicyclic sugar moieties are further
defined by isomeric configuration. For example, an LNA nucleoside
(described herein) may be in the .alpha.-L configuration or in the
.beta.-D configuration.
##STR00002##
.alpha.-L-methyleneoxy (4'-CH.sub.2--O--2') or .alpha.-L-LNA
bicyclic nucleosides have been incorporated into antisense
oligonucleotides that showed antisense activity (Frieden et al.,
Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general
descriptions of bicyclic nucleosides include both isomeric
configurations. When the positions of specific bicyclic nucleosides
(e.g., LNA) are identified in exemplified embodiments herein, they
are in the .beta.-D configuration, unless otherwise specified.
[0280] In certain embodiments, modified sugar moieties comprise one
or more non-bridging sugar substituent and one or more bridging
sugar substituent (e.g., 5'-substituted and 4'-2' bridged
sugars).
[0281] Nucleosides comprising modified furanosyl sugar moieties and
modified furanosyl sugar moieties may be referred to by the
position(s) of the substitution(s) on the sugar moiety of the
nucleoside. The term "modified" following a position of the
furanosyl ring, such as "2'-modified", indicates that the sugar
moiety comprises the indicated modification at the 2' position and
may comprise additional modifications and/or substituents. The term
"substituted" following a position of the furanosyl ring, such as
"2'-substituted" or "2'-4'-substituted", indicates that is the only
position(s) having a substituent other than those found in
unmodified sugar moieties in oligonucleotides. Accordingly, the
following sugar moieties are represented by the following
formulas.
[0282] In the context of a nucleoside and/or an oligonucleotide, a
non-bicyclic, modified furanosyl sugar moiety is represented by
formula I:
##STR00003##
wherein B is a nucleobase; and L.sub.1 and L.sub.2 are each,
independently, an internucleoside linkage, a terminal group, a
conjugate group, or a hydroxyl group. Among the R groups, at least
one of R.sub.3-7 is not H and/or at least one of R.sub.1 and
R.sub.2 is not H or OH. In a 2'-modified furanosyl sugar moiety, at
least one of R.sub.1 and R.sub.2 is not H or OH and each of
R.sub.3-7 is independently selected from H or a substituent other
than H. In a 4'-modified furanosyl sugar moiety, R.sub.5 is not H
and each of R.sub.1-4, 6, 7 are independently selected from H and a
substituent other than H; and so on for each position of the
furanosyl ring. The stereochemistry is not defined unless otherwise
noted.
[0283] In the context of a nucleoside and/or an oligonucleotide, a
non-bicyclic, modified, substituted fuarnosyl sugar moiety is
represented by formula I, wherein B is a nucleobase; and L1 and L2
are each, independently, an internucleoside linkage, a terminal
group, a conjugate group, or a hydroxyl group. Among the R groups,
either one (and no more than one) of R.sub.3-7 is a substituent
other than H or one of R.sub.1 or R.sub.2 is a substituent other
than H or OH. The stereochemistry is not defined unless otherwise
noted. Examples of non-bicyclic, modified, substituted furanosyl
sugar moieties include 2'-substituted ribosyl, 4'-substituted
ribosyl, and 5'-substituted ribosyl sugar moieties, as well as
substituted 2'-deoxyfuranosyl sugar moieties, such as
4'-substituted 2'-deoxyribosyl and 5'-substituted 2'-deoxyribosyl
sugar moieties.
[0284] In the context of a nucleoside and/or an oligonucleotide, a
2'-substituted ribosyl sugar moiety is represented by formula
II:
##STR00004##
wherein B is a nucleobase; and L.sub.1 and L.sub.2 are each,
independently, an internucleoside linkage, a terminal group, a
conjugate group, or a hydroxyl group. R.sub.1 is a substituent
other than H or OH. The stereochemistry is defined as shown.
[0285] In the context of a nucleoside and/or an oligonucleotide, a
4'-substituted ribosyl sugar moiety is represented by formula
III:
##STR00005##
wherein B is a nucleobase; and L.sub.1 and L.sub.2 are each,
independently, an internucleoside linkage, a terminal group, a
conjugate group, or a hydroxyl group. R.sub.5 is a substituent
other than H. The stereochemistry is defined as shown.
[0286] In the context of a nucleoside and/or an oligonucleotide, a
5'-substituted ribosyl sugar moiety is represented by formula
IV:
##STR00006##
wherein B is a nucleobase; and L.sub.1 and L.sub.2 are each,
independently, an internucleoside linkage, a terminal group, a
conjugate group, or a hydroxyl group. R.sub.6 or R.sub.7 is a
substituent other than H. The stereochemistry is defined as
shown.
[0287] In the context of a nucleoside and/or an oligonucleotide, a
2'-deoxyfuranosyl sugar moiety is represented by formula V:
##STR00007##
wherein B is a nucleobase; and L.sub.1 and L.sub.2 are each,
independently, an internucleoside linkage, a terminal group, a
conjugate group, or a hydroxyl group. Each of R.sub.1-5 are
indepently selected from H and a non-H substituent. If all of
R.sub.1-5 are each H, the sugar moiety is an unsubstituted
2'-deoxyfuranosyl sugar moiety. The stereochemistry is not defined
unless otherwise noted.
[0288] In the context of a nucleoside and/or an oligonucleotide, a
4'-substituted 2'-deoxyribosyl sugar moiety is represented by
formula VI:
##STR00008##
wherein B is a nucleobase; and L.sub.1 and L.sub.2 are each,
independently, an internucleoside linkage, a terminal group, a
conjugate group, or a hydroxyl group. R.sub.3 is a substituent
other than H. The stereochemistry is defined as shown.
[0289] In the context of a nucleoside and/or an oligonucleotide, a
5'-substituted 2'-deoxyribosyl sugar moiety is represented by
formula VII:
##STR00009##
wherein B is a nucleobase; and L.sub.1 and L.sub.2 are each,
independently, an internucleoside linkage, a terminal group, a
conjugate group, or a hydroxyl group. R.sub.4 or R.sub.5 is a
substituent other than H. The stereochemistry is defined as
shown.
[0290] Unsubstituted 2'-deoxyfuranosyl sugar moieties may be
unmodified (.beta.-D-2'-deoxyribosyl) or modified. Examples of
modified, unsubstituted 2'-deoxyfuranosyl sugar moieties include
.beta.-L-2'-deoxyribosyl, .alpha.-L-2'-deoxyribosyl,
.alpha.-D-2'-deoxyribosyl, and .beta.-D-xylosyl sugar moieties. For
example, in the context of a nucleoside and/or an oligonucleotide,
a .beta.-L-2'-deoxyribosyl sugar moiety is represented by formula
VIII:
##STR00010##
wherein B is a nucleobase; and L.sub.1 and L.sub.2 are each,
independently, an internucleoside linkage, a terminal group, a
conjugate group, or a hydroxyl group. The stereochemistry is
defined as shown.
[0291] In certain embodiments, modified sugar moieties are sugar
surrogates. In certain such embodiments, the oxygen atom of the
sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen
atom. In certain such embodiments, such modified sugar moieties
also comprise bridging and/or non-bridging substituents as
described herein. For example, certain sugar surrogates comprise a
4'-sulfur atom and a substitution at the 2'-position (see, e.g.,
Bhat et al., U.S. Pat. No. 7,875,733 and Bhat et al., U.S. Pat. No.
7,939,677) and/or the 5' position.
[0292] In certain embodiments, sugar surrogates comprise rings
having other than 5 atoms. For example, in certain embodiments, a
sugar surrogate comprises a six-membered tetrahydropyran ("THP").
Such tetrahydropyrans may be further modified or substituted.
Nucleosides comprising such modified tetrahydropyrans include but
are not limited to hexitol nucleic acid ("HNA"), altritol nucleic
acid ("ANA"), mannitol nucleic acid ("MNA") (see, e.g., Leumann, C
J. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:
##STR00011##
("F-HNA", see e.g. Swayze et al., U.S. Pat. No. 8,088,904; Swayze
et al., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No.
8,796,437; and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA can
also be referred to as a F-THP or 3'-fluoro tetrahydropyran), and
nucleosides comprising additional modified THP compounds having the
formula:
##STR00012##
wherein, independently, for each of said modified THP
nucleoside:
[0293] Bx is a nucleobase moiety;
[0294] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linkage linking the modified THP nucleoside to the
remainder of an oligonucleotide or one of T.sub.3 and T.sub.4 is an
internucleoside linkage linking the modified THP nucleoside to the
remainder of an oligonucleotide and the other of T.sub.3 and
T.sub.4 is H, a hydroxyl protecting group, a linked conjugate
group, or a 5' or 3'-terminal group; [0295] q.sub.1, q.sub.2,
q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each,
independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted
C.sub.2-C.sub.6 alkynyl; and
[0296] each of R.sub.1 and R.sub.2 is independently selected from
among: hydrogen, halogen, substituted or unsubstituted alkoxy,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1,
OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and
CN, wherein X is O, S or NJ.sub.1, and each J.sub.1, J.sub.2, and
J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.
[0297] In certain embodiments, modified THP nucleosides are
provided wherein q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5,
q.sub.6 and q.sub.7 are each H. In certain embodiments, at least
one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and
q.sub.7 is other than H. In certain embodiments, at least one of
q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is
methyl. In certain embodiments, modified THP nucleosides are
provided wherein one of R.sub.1 and R.sub.2 is F. In certain
embodiments, R.sub.1 is F and R.sub.2 is H, in certain embodiments,
R.sub.1 is methoxy and R.sub.2 is H, and in certain embodiments,
R.sub.1 is methoxyethoxy and R.sub.2 is H.
[0298] In certain embodiments, sugar surrogates comprise rings
having more than 5 atoms and more than one heteroatom. For example,
nucleosides comprising morpholino sugar moieties and their use in
oligonucleotides have been reported (see, e.g., Braasch et al.,
Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Pat.
No. 5,698,685; Summerton et al., U.S. Pat. No. 5,166,315; Summerton
et al., U.S. Pat. No. 5,185,444; and Summerton et al., U.S. Pat.
No. 5,034,506). As used here, the term "morpholino" means a sugar
surrogate having the following structure:
##STR00013##
[0299] In certain embodiments, morpholinos may be modified, for
example by adding or altering various substituent groups from the
above morpholino structure. Such sugar surrogates are refered to
herein as "modifed morpholinos."
[0300] In certain embodiments, sugar surrogates comprise acyclic
moieites. Examples of nucleosides and oligonucleotides comprising
such acyclic sugar surrogates include but are not limited to:
peptide nucleic acid ("PNA"), acyclic butyl nucleic acid (see,
e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and
nucleosides and oligonucleotides described in Manoharan et al.,
WO2011/133876.
[0301] Many other bicyclic and tricyclic sugar and sugar surrogate
ring systems are known in the art that can be used in modified
nucleosides.
[0302] In certain embodiments, modified nucleosides are DNA mimics.
In certain embodiments, a DNA mimic is a sugar surrogate. In
certain embodiments, a DNA mimic is a cycohexenyl or hexitol
nucleic acid. In certain embodiments, a DNA mimic is described in
Figure 1 of Vester, et. al., "Chemically modified oligonucleotides
with efficient RNase H response," Bioorg. Med. Chem. Letters, 2008,
18: 2296-2300, incorporated by reference herein. In certain
embodiments, a DNA mimic nucleoside has a formula selected
from:
##STR00014##
wherein Bx is a heterocyclic base moiety. In certain embodiments, a
DNA mimic is .alpha.,.beta.-constrained nucleic acid (CAN),
2',4'-carbocyclic-LNA, or 2',4'-carbocyclic-ENA. In certain
embodiments, a DNA mimic has a sugar moiety selected from among:
4'-C-hydroxymethyl-2'-deoxyribosyl,
3'-C-hydroxymethyl-2'-deoxyribosyl, 3'-C-hydroxymethyl-arabinosyl,
3'-C-2'-O-arabinosyl, 3'-C-methylene-extended-2'-deoxyxylosyl,
3'-C-methylene-extended-xyolosyl, 3'-C-2'-0-piperazino-arabinosyl.
In certain embodiments, a DNA mimic has a sugar moiety selected
from among: 2'-methylribosyl, 2'-S-methylribosyl, 2'-aminoribosyl,
2'-NH(CH2)-ribosyl, 2'-NH(CH2)2-ribosyl, 2'-CH2-F-ribosyl,
2'-CHF2-ribosyl, 2'-CF3-ribosyl, 2'=CF2 ribosyl, 2'-ethylribosyl,
2'-alkenylribosyl, 2'-alkynylribosyl, 2'-O-4'-C-methyleneribosyl,
2'-cyanoarabinosyl, 2'-chloroarabinosyl, 2'-fluoroarabinosyl,
2'-bromoarabinosyl, 2'-azidoarabinosyl, 2'-methoxyarabinosyl, and
2'-arabinosyl. In certain embodiments, a DNA mimic has a sugar
moiety selected from 4'-methyl-modified deoxyfuranosyl,
4'-F-deoxyfuranosyl, 4'-OMe-deoxyfuranosyl. In certain embodiments,
a DNA mimic has a sugar moiety selected from among:
5'-methyl-2'-.beta.-D-deoxyribosyl,
5'-ethyl-2'-.beta.-D-deoxyribosyl,
5'-allyl-2'-.beta.-D-deoxyribosyl,
2'-fluoro-.beta.-D-arabinofuranosyl. In certain embodiments, DNA
mimics are listed on page 32-33 of PCT/US00/267929 as B-form
nucleotides, incorporated by reference herein in its entirety.
[0303] 2. Modified Nucleobases
[0304] In certain embodiments, modified nucleobases are selected
from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl
substituted pyrimidines, alkyl substituted purines, and N-2, N-6
and O-6 substituted purines. In certain embodiments, modified
nucleobases are selected from: 2-aminopropyladenine,
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-N-methylguanine, 6-N-methyladenine, 2-propyladenine ,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl
(--C.ident.C--CH.sub.3) uracil, 5-propynylcytosine, 6-azouracil,
6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl,
8-aza and other 8-substituted purines, 5-halo, particularly
5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine,
7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine,
7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine,
6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine,
4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl
4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous
bases, size-expanded bases, and fluorinated bases. Further modified
nucleobases include tricyclic pyrimidines, such as
1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and
9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified
nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in Merigan et al., U.S.
Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of
Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley
& Sons, 1990, 858-859; Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15,
Antisense Research and Applications, Crooke, S. T. and Lebleu, B.,
Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6
and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press,
2008, 163-166 and 442-443.
[0305] Publications that teach the preparation of certain of the
above noted modified nucleobases as well as other modified
nucleobases include without limitation, Manoharan et al.,
US2003/0158403; Manoharan et al., US2003/0175906; Dinh et al., U.S.
Pat. No. 4,845,205; Spielvogel et al., U.S. Pat. No. 5,130,302;
Rogers et al., U.S. Pat. No. 5,134,066; Bischofberger et al., U.S.
Pat. No. 5,175,273; Urdea et al., U.S. Pat. No. 5,367,066; Benner
et al., U.S. Pat. No. 5,432,272; Matteucci et al., U.S. Pat. No.
5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cook et al.,
U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No. 5,484,908;
Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al., U.S.
Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540;
Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat.
No. 5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et
al., U.S. Pat. No. 5,614,617; Froehler et al., U.S. Pat. No.
5,645,985; Cook et al., U.S. Pat. No. 5,681,941; Cook et al., U.S.
Pat. No. 5,811,534; Cook et al., U.S. Pat. No. 5,750,692; Cook et
al., U.S. Pat. No. 5,948,903; Cook et al., U.S. Pat. No. 5,587,470;
Cook et al., U.S. Pat. No. 5,457,191; Matteucci et al., U.S. Pat.
No. 5,763,588; Froehler et al., U.S. Pat. No. 5,830,653; Cook et
al., U.S. Pat. No. 5,808,027; Cook et al., 6,166,199; and Matteucci
et al., U.S. Pat. No. 6,005,096.
[0306] In certain embodiments, compounds comprise or consist of a
modified oligonucleotide complementary to an target nucleic acid
comprising one or more modified nucleobases. In certain
embodiments, the modified nucleobase is 5-methylcytosine. In
certain embodiments, each cytosine is a 5-methylcyto sine.
B. Modified Internucleoside Linkages
[0307] In certain embodiments, compounds described herein having
one or more modified internucleoside linkages are selected over
compounds having only phosphodiester internucleoside linkages
because of desirable properties such as, for example, enhanced
cellular uptake, enhanced affinity for target nucleic acids, and
increased stability in the presence of nucleases.
[0308] In certain embodiments, compounds comprise or consist of a
modified oligonucleotide complementary to a target nucleic acid
comprising one or more modified internucleoside linkages. In
certain embodiments, the modified internucleoside linkages are
phosphorothioate linkages. In certain embodiments, each
internucleoside linkage of an antisense compound is a
phosphorothioate internucleoside linkage.
[0309] In certain embodiments, nucleosides of modified
oligonucleotides may be linked together using any internucleoside
linkage. The two main classes of internucleoside linkages are
defined by the presence or absence of a phosphorus atom.
Representative phosphorus-containing internucleoside linkages
include unmodified phosphodiester internucleoside linkages,
modified phosphotriesters such as THP phosphotriester and isopropyl
phosphotriester, phosphonates such as methylphosphonate, isopropyl
phosphonate, isobutyl phosphonate, and phosphonoacetate,
phosphoramidates, phosphorothioate, and phosphorodithioate
("HS--P.dbd.S"). Representative non-phosphorus containing
internucleoside linkages include but are not limited to
methylenemethylimino (--CH.sub.2--N(CH.sub.3)--O--CH2--),
thiodiester, thionocarbamate (--O--C(.dbd.O)(NH)--S--); siloxane
(--O--SiH.sub.2--O--); formacetal, thioacetamido (TANA),
alt-thioformacetal, glycine amide, and N,N'-dimethylhydrazine
(--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). Modified internucleoside
linkages, compared to naturally occurring phosphate linkages, can
be used to alter, typically increase, nuclease resistance of the
oligonucleotide. Methods of preparation of phosphorous-containing
and non-phosphorous-containing internucleoside linkages are well
known to those skilled in the art.
[0310] Representative internucleoside linkages having a chiral
center include but are not limited to alkylphosphonates and
phosphorothioates. Modified oligonucleotides comprising
internucleoside linkages having a chiral center can be prepared as
populations of modified oligonucleotides comprising stereorandom
internucleoside linkages, or as populations of modified
oligonucleotides comprising phosphorothioate linkages in particular
stereochemical configurations. In certain embodiments, populations
of modified oligonucleotides comprise phosphorothioate
internucleoside linkages wherein all of the phosphorothioate
internucleoside linkages are stereorandom. Such modified
oligonucleotides can be generated using synthetic methods that
result in random selection of the stereochemical configuration of
each phosphorothioate linkage. Nonetheless, as is well understood
by those of skill in the art, each individual phosphorothioate of
each individual oligonucleotide molecule has a defined
stereoconfiguration. In certain embodiments, populations of
modified oligonucleotides are enriched for modified
oligonucleotides comprising one or more particular phosphorothioate
internucleoside linkages in a particular, independently selected
stereochemical configuration. In certain embodiments, the
particular configuration of the particular phosphorothioate linkage
is present in at least 65% of the molecules in the population. In
certain embodiments, the particular configuration of the particular
phosphorothioate linkage is present in at least 70% of the
molecules in the population. In certain embodiments, the particular
configuration of the particular phosphorothioate linkage is present
in at least 80% of the molecules in the population. In certain
embodiments, the particular configuration of the particular
phosphorothioate linkage is present in at least 90% of the
molecules in the population. In certain embodiments, the particular
configuration of the particular phosphorothioate linkage is present
in at least 99% of the molecules in the population. Such chirally
enriched populations of modified oligonucleotides can be generated
using synthetic methods known in the art, e.g., methods described
in Oka et al., JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res.
42, 13456 (2014), and WO 2017/015555. In certain embodiments, a
population of modified oligonucleotides is enriched for modified
oligonucleotides having at least one indicated phosphorothioate in
the (Sp) configuration. In certain embodiments, a population of
modified oligonucleotides is enriched for modified oligonucleotides
having at least one phosphorothioate in the (Rp) configuration. In
certain embodiments, modified oligonucleotides comprising (Rp)
and/or (Sp) phosphorothioates comprise one or more of the following
formulas, respectively, wherein "B" indicates a nucleobase:
##STR00015##
Unless otherwise indicated, chiral internucleoside linkages of
modified oligonucleotides described herein can be stereorandom or
in a particular stereochemical configuration.
[0311] Neutral internucleoside linkages include, without
limitation, phosphotriesters, phosphonates, MMI
(3'-CH.sub.2--N(CH.sub.3)--O-5'), amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5'), amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5'), formacetal (3'-O--CH.sub.2-O-5),
methoxypropyl, and thioformacetal (3'-S--CH.sub.2--O-5'). Further
neutral internucleoside linkages include nonionic linkages
comprising siloxane (dialkylsiloxane), carboxylate ester,
carboxamide, sulfide, sulfonate ester and amides (See for example:
Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and
P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4,
40-65). Further neutral internucleoside linkages include nonionic
linkages comprising mixed N, O, S and CH2 component parts.
[0312] II. Certain Motifs
[0313] In certain embodiments, oligomeric compounds described
herein comprise or consist of oligonucleotides. Oligonucleotides
can have a motif, e.g. a pattern of unmodified and/or modified
sugar moieties, nucleobases, and/or internucleoside linkages. In
certain embodiments, modified oligonucleotides comprise one or more
modified nucleoside comprising a modified sugar. In certain
embodiments, modified oligonucleotides comprise one or more
modified nucleosides comprising a modified nucleobase. In certain
embodiments, modified oligonucleotides comprise one or more
modified internucleoside linkage. In such embodiments, the
modified, unmodified, and differently modified sugar moieties,
nucleobases, and/or internucleoside linkages of a modified
oligonucleotide define a pattern or motif. In certain embodiments,
the patterns or motifs of sugar moieties, nucleobases, and
internucleoside linkages are each independent of one another. Thus,
a modified oligonucleotide may be described by its sugar motif,
nucleobase motif and/or internucleoside linkage motif (as used
herein, nucleobase motif describes the modifications to the
nucleobases independent of the sequence of nucleobases).
[0314] A. Certain Sugar Motifs
[0315] In certain embodiments, oligomeric compounds described
herein comprise or consist of oligonucleotides. In certain
embodiments, oligonucleotides comprise one or more type of modified
sugar and/or unmodified sugar moiety arranged along the
oligonucleotide or region thereof in a defined pattern or sugar
motif. In certain instances, such sugar motifs include but are not
limited to any of the sugar modifications discussed herein.
[0316] In certain embodiments, a modified oligonucleotide comprises
or has a uniformly modified sugar motif. An oligonucleotide
comprising a uniformly modified sugar motif comprises a segment of
linked nucleosides, wherein each nucleoside of the segment
comprises the same modified sugar moiety. An oligonucleotide having
a uniformly modified sugar motif throughout the entirety of the
oligonucleotide comprises only nucleosides comprising the same
modified sugar moiety. For example, each nucleoside of a 2'-MOE
uniformly modified oligonucleotide comprises a 2'-MOE modified
sugar moiety. An oligonucleotide comprising or having a uniformly
modified sugar motif can have any nucleobase sequence and any
internucleoside linkage motif
[0317] B. Certain Nucleobase Motifs
[0318] In certain embodiments, oligomeric compounds described
herein comprise or consist of oligonucleotides. In certain
embodiments, oligonucleotides comprise modified and/or unmodified
nucleobases arranged along the oligonucleotide or region thereof in
a defined pattern or motif. In certain embodiments, each nucleobase
is modified. In certain embodiments, none of the nucleobases are
modified. In certain embodiments, each purine or each pyrimidine is
modified. In certain embodiments, each adenine is modified. In
certain embodiments, each guanine is modified. In certain
embodiments, each thymine is modified. In certain embodiments, each
uracil is modified. In certain embodiments, each cytosine is
modified. In certain embodiments, some or all of the cytosine
nucleobases in a modified oligonucleotide are
5-methylcytosines.
[0319] In certain embodiments, modified oligonucleotides comprise a
block of modified nucleobases. In certain such embodiments, the
block is at the 3'-end of the oligonucleotide. In certain
embodiments the block is within 3 nucleosides of the 3'-end of the
oligonucleotide. In certain embodiments, the block is at the 5'-end
of the oligonucleotide. In certain embodiments the block is within
3 nucleosides of the 5'-end of the oligonucleotide.
[0320] C. Certain Internucleoside Linkage Motifs
[0321] In certain embodiments, oligomeric compounds described
herein comprise or consist of oligonucleotides. In certain
embodiments, oligonucleotides comprise modified and/or unmodified
internucleoside linkages arranged along the oligonucleotide or
region thereof in a defined pattern or motif. In certain
embodiments, each internucleoside linkage is a phosphodiester
internucleoside linkage (P.dbd.O). In certain embodiments, each
internucleoside linkage of a modified oligonucleotide is a
phosphorothioate internucleoside linkage (P.dbd.S). In certain
embodiments, each internucleoside linkage of a modified
oligonucleotide is independently selected from a phosphorothioate
internucleoside linkage and phosphodiester internucleoside linkage.
In certain embodiments, each phosphorothioate internucleoside
linkage is independently selected from a stereorandom
phosphorothioate, a (Sp) phosphorothioate, and a (Rp)
phosphorothioate. In certain embodiments, the terminal
internucleoside linkages are modified. In certain embodiments, the
internucleoside linkage motif comprises at least one phosphodiester
internucleoside linkage in at least one of the 5'-region and the
3'-region, wherein the at least one phosphodiester linkage is not a
terminal internucleoside linkage, and the remaining internucleoside
linkages are phosphorothioate internucleoside linkages. In certain
such embodiments, all of the phosphorothioate linkages are
stereorandom. In certain embodiments, populations of modified
oligonucleotides are enriched for modified oligonucleotides
comprising such internucleoside linkage motifs.
[0322] In certain embodiments, oligonucleotides comprise a region
having an alternating internucleoside linkage motif. In certain
embodiments, oligonucleotides comprise a region of uniformly
modified internucleoside linkages. In certain such embodiments, the
internucleoside linkages are phosphorothioate internucleoside
linkages. In certain embodiments, all of the internucleoside
linkages of the oligonucleotide are phosphorothioate
internucleoside linkages. In certain embodiments, each
internucleoside linkage of the oligonucleotide is selected from
phosphodiester or phosphate and phosphorothioate. In certain
embodiments, each internucleoside linkage of the oligonucleotide is
selected from phosphodiester or phosphate and phosphorothioate and
at least one internucleoside linkage is phosphorothioate.
[0323] In certain embodiments, the oligonucleotide comprises at
least 6 phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least 8
phosphorothioate internucleoside linkages. In certain embodiments,
the oligonucleotide comprises at least 10 phosphorothioate
internucleoside linkages. In certain embodiments, the
oligonucleotide comprises at least one block of at least 6
consecutive phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least one block of at
least 8 consecutive phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide comprises at least one
block of at least 10 consecutive phosphorothioate internucleoside
linkages. In certain embodiments, the oligonucleotide comprises at
least block of at least one 12 consecutive phosphorothioate
internucleoside linkages. In certain such embodiments, at least one
such block is located at the 3' end of the oligonucleotide. In
certain such embodiments, at least one such block is located within
3 nucleosides of the 3' end of the oligonucleotide.
[0324] In certain embodiments, oligonucleotides comprise one or
more methylphosphonate linkages. In certain embodiments, modified
oligonucleotides comprise a linkage motif comprising all
phosphorothioate linkages except for one or two methylphosphonate
linkages.
[0325] In certain embodiments, it is desirable to arrange the
number of phosphorothioate internucleoside linkages and
phosphodiester internucleoside linkages to maintain nuclease
resistance. In certain embodiments, it is desirable to arrange the
number and position of phosphorothioate internucleoside linkages
and the number and position of phosphodiester internucleoside
linkages to maintain nuclease resistance. In certain embodiments,
the number of phosphorothioate internucleoside linkages may be
decreased and the number of phosphodiester internucleoside linkages
may be increased. In certain embodiments, the number of
phosphorothioate internucleoside linkages may be decreased and the
number of phosphodiester internucleoside linkages may be increased
while still maintaining nuclease resistance. In certain embodiments
it is desirable to decrease the number of phosphorothioate
internucleoside linkages while retaining nuclease resistance. In
certain embodiments it is desirable to increase the number of
phosphodiester internucleoside linkages while retaining nuclease
resistance.
[0326] III. Certain Modified Oligonucleotides
[0327] In certain embodiments, oligomeric compounds described
herein comprise or consist of modified oligonucleotides. In certain
embodiments, the above modifications (sugar, nucleobase,
internucleoside linkage) are incorporated into a modified
oligonucleotide. In certain embodiments, modified oligonucleotides
are characterized by their modifications, motifs, and overall
lengths. In certain embodiments, such parameters are each
independent of one another. Thus, unless otherwise indicated, each
internucleoside linkage of a modified oligonucleotide may be
modified or unmodified and may or may not follow the modification
pattern of the sugar moieties. Likewise, such modified
oligonucleotides may comprise one or more modified nucleobase
independent of the pattern of the sugar modifications. Furthermore,
in certain instances, a modified oligonucleotide is described by an
overall length or range and by lengths or length ranges of two or
more regions (e.g., a region of nucleosides having specified sugar
modifications), in such circumstances it may be possible to select
numbers for each range that result in an oligonucleotide having an
overall length falling outside the specified range. In such
circumstances, both elements must be satisfied. For example, in
certain embodiments, a modified oligonucleotide consists of 15-20
linked nucleosides and has a sugar motif consisting of three
regions or segments, A, B, and C, wherein region or segment A
consists of 2-6 linked nucleosides having a specified sugar motif,
region or segment B consists of 6-10 linked nucleosides having a
specified sugar motif, and region or segment C consists of 2-6
linked nucleosides having a specified sugar motif. Such embodiments
do not include modified oligonucleotides where A and C each consist
of 6 linked nucleosides and B consists of 10 linked nucleosides
(even though those numbers of nucleosides are permitted within the
requirements for A, B, and C) because the overall length of such
oligonucleotide is 22, which exceeds the upper limit of 20 for the
overall length of the modified oligonucleotide. Unless otherwise
indicated, all modifications are independent of nucleobase sequence
except that the modified nucleobase 5-methylcytosine is necessarily
a "C" in an oligonucleotide sequence.
[0328] In certain embodiments, oligonucleotides consist of X to Y
linked nucleosides, where X represents the fewest number of
nucleosides in the range and Y represents the largest number
nucleosides in the range. In certain such embodiments, X and Y are
each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and
50; provided that X.ltoreq.Y. For example, in certain embodiments,
oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16,
12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to
23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12
to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19,
13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to
26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14
to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23,
14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to
30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15
to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28,
15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to
21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16
to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21,
17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to
28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18
to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29,
18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to
25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20
to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28,
20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to
26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22
to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24,
23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to
25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25
to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29,
26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29
to 30 linked nucleosides.
[0329] In certain embodiments oligonucleotides have a nucleobase
sequence that is complementary to a second oligonucleotide or an
identified reference nucleic acid, such as a target nucleic acid.
In certain embodiments, a region of an oligonucleotide has a
nucleobase sequence that is complementary to a second
oligonucleotide or an identified reference nucleic acid, such as a
target nucleic acid. In certain embodiments, the nucleobase
sequence of a region or entire length of an oligonucleotide is at
least 70%, at least 80%, at least 90%, at least 95%, or 100%
complementary to the second oligonucleotide or nucleic acid, such
as a target nucleic acid.
[0330] IV. Certain Conjugated Compounds
[0331] In certain embodiments, the oligomeric compounds described
herein comprise or consist of an oligonucleotide (modified or
unmodified) and optionally one or more conjugate groups and/or
terminal groups. Conjugate groups consist of one or more conjugate
moiety and a conjugate linker that links the conjugate moiety to
the oligonucleotide. Conjugate groups may be attached to either or
both ends of an oligonucleotide and/or at any internal position. In
certain embodiments, conjugate groups are attached to the
2'-position of a nucleoside of a modified oligonucleotide. In
certain embodiments, conjugate groups that are attached to either
or both ends of an oligonucleotide are terminal groups. In certain
such embodiments, conjugate groups or terminal groups are attached
at the 3' and/or 5'-end of oligonucleotides. In certain such
embodiments, conjugate groups (or terminal groups) are attached at
the 3'-end of oligonucleotides. In certain embodiments, conjugate
groups are attached near the 3'-end of oligonucleotides. In certain
embodiments, conjugate groups (or terminal groups) are attached at
the 5'-end of oligonucleotides. In certain embodiments, conjugate
groups are attached near the 5'-end of oligonucleotides.
[0332] Examples of terminal groups include but are not limited to
conjugate groups, capping groups, phosphate moieties, protecting
groups, modified or unmodified nucleosides, and two or more
nucleosides that are independently modified or unmodified.
[0333] A. Certain Conjugate Groups
[0334] In certain embodiments, oligonucleotides are covalently
attached to one or more conjugate groups. In certain embodiments,
conjugate groups modify one or more properties of the attached
oligonucleotide, including but not limited to pharmacodynamics,
pharmacokinetics, stability, binding, absorption, tissue
distribution, cellular distribution, cellular uptake, charge and
clearance. In certain embodiments, conjugate groups impart a new
property on the attached oligonucleotide, e.g., fluorophores or
reporter groups that enable detection of the oligonucleotide.
[0335] Certain conjugate groups and conjugate moieties have been
described previously, for example: cholesterol moiety (Letsinger et
al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid
(Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a
thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.
Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et
al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain,
e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al.,
EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,
327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic, a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, i, 923-937),.sub.=a tocopherol group
(Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220;
doi:10.1038/mtna.2014.72 and Nishina et al., Molecular Therapy,
2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/179620).
[0336] 1. Conjugate Moieties
[0337] Conjugate moieties include, without limitation,
intercalators, reporter molecules, polyamines, polyamides,
peptides, carbohydrates (e.g., GalNAc), vitamin moieties,
polyethylene glycols, thioethers, polyethers, cholesterols,
thiocholesterols, cholic acid moieties, folate, lipids,
phospholipids, biotin, phenazine, phenanthridine, anthraquinone,
adamantane, acridine, fluoresceins, rhodamines, coumarins,
fluorophores, and dyes.
[0338] In certain embodiments, a conjugate moiety comprises an
active drug substance, for example, aspirin, warfarin,
phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen,
(S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic
acid, a benzothiadiazide, chlorothiazide, a diazepine,
indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
[0339] 2. Conjugate Linkers
[0340] Conjugate moieties are attached to oligonucleotides through
conjugate linkers. In certain oligomeric compounds, a conjugate
linker is a single chemical bond (i.e. conjugate moiety is attached
to an oligonucleotide via a conjugate linker through a single
bond). In certain embodiments, the conjugate linker comprises a
chain structure, such as a hydrocarbyl chain, or an oligomer of
repeating units such as ethylene glycol, nucleosides, or amino acid
units.
[0341] In certain embodiments, a conjugate linker comprises one or
more groups selected from alkyl, amino, oxo, amide, disulfide,
polyethylene glycol, ether, thioether, and hydroxylamino. In
certain such embodiments, the conjugate linker comprises groups
selected from alkyl, amino, oxo, amide and ether groups. In certain
embodiments, the conjugate linker comprises groups selected from
alkyl and amide groups. In certain embodiments, the conjugate
linker comprises groups selected from alkyl and ether groups. In
certain embodiments, the conjugate linker comprises at least one
phosphorus moiety. In certain embodiments, the conjugate linker
comprises at least one phosphate group. In certain embodiments, the
conjugate linker includes at least one neutral linking group.
[0342] In certain embodiments, conjugate linkers, including the
conjugate linkers described above, are bifunctional linking
moieties, e.g., those known in the art to be useful for attaching
conjugate groups to oligomeric compounds, such as the
oligonucleotides provided herein. In general, a bifunctional
linking moiety comprises at least two functional groups. One of the
functional groups is selected to bind to a particular site on an
oligomeric compound and the other is selected to bind to a
conjugate group. Examples of functional groups used in a
bifunctional linking moiety include but are not limited to
electrophiles for reacting with nucleophilic groups and
nucleophiles for reacting with electrophilic groups. In certain
embodiments, bifunctional linking moieties comprise one or more
groups selected from amino, hydroxyl, carboxylic acid, thiol,
alkyl, alkenyl, and alkynyl.
[0343] Examples of conjugate linkers include but are not limited to
pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl
4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC) and
6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include
but are not limited to substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl or substituted or unsubstituted
C.sub.2-C.sub.10 alkynyl, wherein a nonlimiting list of preferred
substituent groups includes hydroxyl, amino, alkoxy, carboxy,
benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,
alkenyl and alkynyl.
[0344] In certain embodiments, conjugate linkers comprise 1-10
linker-nucleosides. In certain embodiments, such linker-nucleosides
are modified nucleosides. In certain embodiments such
linker-nucleosides comprise a modified sugar moiety. In certain
embodiments, linker-nucleosides are unmodified. In certain
embodiments, linker-nucleosides comprise an optionally protected
heterocyclic base selected from a purine, substituted purine,
pyrimidine or substituted pyrimidine. In certain embodiments, a
cleavable moiety is a nucleoside selected from uracil, thymine,
cytosine, 4-N-benzoylcytosine, 5-methylcytosine,
4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine
and 2-N-isobutyrylguanine. It is typically desirable for
linker-nucleosides to be cleaved from the oligomeric compound after
it reaches a target tissue. Accordingly, linker-nucleosides are
typically linked to one another and to the remainder of the
oligomeric compound through cleavable bonds. In certain
embodiments, such cleavable bonds are phosphodiester bonds.
[0345] Herein, linker-nucleosides are not considered to be part of
the oligonucleotide. Accordingly, in embodiments in which an
oligomeric compound comprises an oligonucleotide consisting of a
specified number or range of linked nucleosides and/or a specified
percent complementarity to a reference nucleic acid and the
oligomeric compound also comprises a conjugate group comprising a
conjugate linker comprising linker-nucleosides, those
linker-nucleosides are not counted toward the length of the
oligonucleotide and are not used in determining the percent
complementarity of the oligonucleotide for the reference nucleic
acid. For example, an oligomeric compound may comprise (1) a
modified oligonucleotide consisting of 8-30 nucleosides and (2) a
conjugate group comprising 1-10 linker-nucleosides that are
contiguous with the nucleosides of the modified oligonucleotide.
The total number of contiguous linked nucleosides in such a
compound is more than 30. Alternatively, an oligomeric compound may
comprise a modified oligonucleotide consisting of 8-30 nucleosides
and no conjugate group. The total number of contiguous linked
nucleosides in such a compound is no more than 30. Unless otherwise
indicated conjugate linkers comprise no more than 10
linker-nucleosides. In certain embodiments, conjugate linkers
comprise no more than 5 linker-nucleosides. In certain embodiments,
conjugate linkers comprise no more than 3 linker-nucleosides. In
certain embodiments, conjugate linkers comprise no more than 2
linker-nucleosides. In certain embodiments, conjugate linkers
comprise no more than 1 linker-nucleoside.
[0346] In certain embodiments, it is desirable for a conjugate
group to be cleaved from the oligonucleotide. For example, in
certain circumstances oligomeric compounds comprising a particular
conjugate moiety are better taken up by a particular cell type, but
once the compound has been taken up, it is desirable that the
conjugate group be cleaved to release the unconjugated
oligonucleotide. Thus, certain conjugate may comprise one or more
cleavable moieties, typically within the conjugate linker. In
certain embodiments, a cleavable moiety is a cleavable bond. In
certain embodiments, a cleavable moiety is a group of atoms
comprising at least one cleavable bond. In certain embodiments, a
cleavable moiety comprises a group of atoms having one, two, three,
four, or more than four cleavable bonds. In certain embodiments, a
cleavable moiety is selectively cleaved inside a cell or
subcellular compartment, such as a lysosome. In certain
embodiments, a cleavable moiety is selectively cleaved by
endogenous enzymes, such as nucleases.
[0347] In certain embodiments, a cleavable bond is selected from
among: an amide, an ester, an ether, one or both esters of a
phosphodiester, a phosphate ester, a carbamate, or a disulfide. In
certain embodiments, a cleavable bond is one or both of the esters
of a phosphodiester. In certain embodiments, a cleavable moiety
comprises a phosphate or phosphodiester. In certain embodiments,
the cleavable moiety is a phosphate linkage between an
oligonucleotide and a conjugate moiety or conjugate group.
[0348] In certain embodiments, a cleavable moiety comprises or
consists of one or more linker-nucleosides. In certain such
embodiments, one or more linker-nucleosides are linked to one
another and/or to the remainder of the oligomeric compound through
cleavable bonds. In certain embodiments, such cleavable bonds are
unmodified phosphodiester bonds. In certain embodiments, a
cleavable moiety is 2'-deoxy nucleoside that is attached to either
the 3' or 5'-terminal nucleoside of an oligonucleotide by a
phosphate internucleoside linkage and covalently attached to the
remainder of the conjugate linker or conjugate moiety by a
phosphate or phosphorothioate linkage. In certain such embodiments,
the cleavable moiety is 2'-deoxyadenosine.
[0349] 3. Certain Cell-Targeting Conjugate Moieties
[0350] In certain embodiments, a conjugate group comprises a
cell-targeting conjugate moiety. In certain embodiments, a
conjugate group has the general formula:
##STR00016##
[0351] wherein n is from 1 to about 3, m is 0 when n is 1, m is 1
when n is 2 or greater, j is 1 or 0, and k is 1 or 0.
[0352] In certain embodiments, n is 1, j is 1 and k is 0. In
certain embodiments, n is 1, j is 0 and k is 1. In certain
embodiments, n is 1, j is 1 and k is 1. In certain embodiments, n
is 2, j is 1 and k is 0. In certain embodiments, n is 2, j is 0 and
k is 1. In certain embodiments, n is 2, j is 1 and k is 1. In
certain embodiments, n is 3, j is 1 and k is 0. In certain
embodiments, n is 3, j is 0 and k is 1. In certain embodiments, n
is 3, j is 1 and k is 1.
[0353] In certain embodiments, conjugate groups comprise
cell-targeting moieties that have at least one tethered ligand. In
certain embodiments, cell-targeting moieties comprise two tethered
ligands covalently attached to a branching group. In certain
embodiments, cell-targeting moieties comprise three tethered
ligands covalently attached to a branching group.
[0354] In certain embodiments, the cell-targeting moiety comprises
a branching group comprising one or more groups selected from
alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether,
thioether and hydroxylamino groups. In certain embodiments, the
branching group comprises a branched aliphatic group comprising
groups selected from alkyl, amino, oxo, amide, disulfide,
polyethylene glycol, ether, thioether and hydroxylamino groups. In
certain such embodiments, the branched aliphatic group comprises
groups selected from alkyl, amino, oxo, amide and ether groups. In
certain such embodiments, the branched aliphatic group comprises
groups selected from alkyl, amino and ether groups. In certain such
embodiments, the branched aliphatic group comprises groups selected
from alkyl and ether groups. In certain embodiments, the branching
group comprises a mono or polycyclic ring system.
[0355] In certain embodiments, each tether of a cell-targeting
moiety comprises one or more groups selected from alkyl,
substituted alkyl, ether, thioether, disulfide, amino, oxo, amide,
phosphodiester, and polyethylene glycol, in any combination. In
certain embodiments, each tether is a linear aliphatic group
comprising one or more groups selected from alkyl, ether,
thioether, disulfide, amino, oxo, amide, and polyethylene glycol,
in any combination. In certain embodiments, each tether is a linear
aliphatic group comprising one or more groups selected from alkyl,
phosphodiester, ether, amino, oxo, and amide, in any combination.
In certain embodiments, each tether is a linear aliphatic group
comprising one or more groups selected from alkyl, ether, amino,
oxo, and amid, in any combination. In certain embodiments, each
tether is a linear aliphatic group comprising one or more groups
selected from alkyl, amino, and oxo, in any combination. In certain
embodiments, each tether is a linear aliphatic group comprising one
or more groups selected from alkyl and oxo, in any combination. In
certain embodiments, each tether is a linear aliphatic group
comprising one or more groups selected from alkyl and
phosphodiester, in any combination. In certain embodiments, each
tether comprises at least one phosphorus linking group or neutral
linking group. In certain embodiments, each tether comprises a
chain from about 6 to about 20 atoms in length. In certain
embodiments, each tether comprises a chain from about 10 to about
18 atoms in length. In certain embodiments, each tether comprises
about 10 atoms in chain length.
[0356] In certain embodiments, each ligand of a cell-targeting
moiety has an affinity for at least one type of receptor on a
target cell. In certain embodiments, each ligand has an affinity
for at least one type of receptor on the surface of a mammalian
lung cell.
[0357] In certain embodiments, each ligand of a cell-targeting
moiety is a carbohydrate, carbohydrate derivative, modified
carbohydrate, polysaccharide, modified polysaccharide, or
polysaccharide derivative. In certain such embodiments, the
conjugate group comprises a carbohydrate cluster (see, e.g., Maier
et al., "Synthesis of Antisense Oligonucleotides Conjugated to a
Multivalent Carbohydrate Cluster for Cellular Targeting,"
Bioconjugate Chemistry, 2003, 14, 18-29, or Rensen et al., "Design
and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids
for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein
Receptor," J. Med. Chem. 2004, 47, 5798-5808, which are
incorporated herein by reference in their entirety). In certain
such embodiments, each ligand is an amino sugar or a thio sugar.
For example, amino sugars may be selected from any number of
compounds known in the art, such as sialic acid,
.alpha.-D-galactosamine, .beta.-muramic acid,
2-deoxy-2-methylamino-L-glucopyranose,
4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,
2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and
N-glycoloyl-.alpha.-neuraminic acid. For example, thio sugars may
be selected from 5-Thio-.beta.-D-glucopyranose, methyl
2,3,4-tri-O-acetyl-1-thio-6-O-trityl-.alpha.-D-glucopyranoside,
4-thio-.beta.-D-galactopyranose, and ethyl
3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-.alpha.-D-gluco-heptopyranoside-
.
[0358] In certain embodiments, oligomeric compounds described
herein comprise a conjugate group found in any of the following
references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al.,
J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein
Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261;
Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al.,
Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem,
1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53,
759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et
al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol,
2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276,
37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43;
Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al.,
Bioorg Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et al.,
Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med
Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011, 19,
2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46;
Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448;
Biessen et al., J Med Chem, 1995, 38, 1846-1852; Sliedregt et al.,
J Med Chem, 1999, 42, 609-618; Rensen et al., J Med Chem, 2004, 47,
5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26,
169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato
et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org
Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14,
1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et
al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug
Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12,
5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12,
103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et
al., Bioorg Med Chem, 2013, 21, 5275-5281; International
applications WO1998/013381; WO2011/038356; WO1997/046098;
WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053;
WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230;
WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607;
WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563;
WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187;
WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352;
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4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;
8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;
6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;
8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;
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US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044;
US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869;
US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042;
US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115;
US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509;
US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512;
US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and
US2009/0203132.
Compositions and Methods for Formulating Pharmaceutical
Compositions
[0359] Oligomeric compounds described herein may be admixed with
pharmaceutically acceptable active or inert substances for the
preparation of pharmaceutical compositions. Compositions and
methods for the formulation of pharmaceutical compositions are
dependent upon a number of criteria, including, but not limited to,
route of administration, extent of disease, or dose to be
administered.
[0360] Certain embodiments provide pharmaceutical compositions
comprising one or more oligomeric compounds or a salt thereof. In
certain embodiments, the oligomeric compounds comprise or consist
of a modified oligonucleotide. In certain such embodiments, the
pharmaceutical composition comprises a suitable pharmaceutically
acceptable diluent or carrier. In certain embodiments, a
pharmaceutical composition comprises a sterile saline solution and
one or more oligomeric compound. In certain embodiments, such
pharmaceutical composition consists of a sterile saline solution
and one or more oligomeric compound. In certain embodiments, the
sterile saline is pharmaceutical grade saline. In certain
embodiments, a pharmaceutical composition comprises one or more
oligomeric compound and sterile water. In certain embodiments, a
pharmaceutical composition consists of one oligomeric compound and
sterile water. In certain embodiments, the sterile water is
pharmaceutical grade water. In certain embodiments, a
pharmaceutical composition comprises or consists of one or more
oligomeric compound and phosphate-buffered saline (PBS). In certain
embodiments, a pharmaceutical composition consists of one or more
oligomeric compound and sterile PBS. In certain embodiments, the
sterile PBS is pharmaceutical grade PBS. Compositions and methods
for the formulation of pharmaceutical compositions are dependent
upon a number of criteria, including, but not limited to, route of
administration, extent of disease, or dose to be administered.
[0361] An oligomeric compound described herein complementary to a
target nucleic acid can be utilized in pharmaceutical compositions
by combining the oligomeric compound with a suitable
pharmaceutically acceptable diluent or carrier and/or additional
components such that the pharmaceutical composition is suitable for
injection. In certain embodiments, a pharmaceutically acceptable
diluent is phosphate buffered saline. Accordingly, in one
embodiment, employed in the methods described herein is a
pharmaceutical composition comprising an oligomeric compound
complementary to a target nucleic acid and a pharmaceutically
acceptable diluent. In certain embodiments, the pharmaceutically
acceptable diluent is phosphate buffered saline. In certain
embodiments, the oligomeric compound comprises or consists of a
modified oligonucleotide provided herein.
[0362] Pharmaceutical compositions comprising oligomeric compounds
provided herein encompass any pharmaceutically acceptable salts,
esters, or salts of such esters, or any other oligonucleotide
which, upon administration to an animal, including a human, is
capable of providing (directly or indirectly) the biologically
active metabolite or residue thereof. In certain embodiments, the
oligomeric compound comprises or consists of a modified
oligonucleotide. Accordingly, for example, the disclosure is also
drawn to pharmaceutically acceptable salts of compounds, prodrugs,
pharmaceutically acceptable salts of such prodrugs, and other
bioequivalents. Suitable pharmaceutically acceptable salts include,
but are not limited to, sodium and potassium salts.
Certain Mechanisms
[0363] Site specific introduction of (Sp) or (Rp) phosphorothioate
internucleoside linkages into the central region of a modified
oligonucleotide can alter the protein binding properties of the
modified oligonucleotide.
[0364] In certain embodiments, site specific introduction of (Sp)
or (Rp) phosphorothioate internucleoside linkages into the central
region of a modified oligonucleotide can alter the cleavage pattern
of RNase H1 when the modified oligonucleotide binds to a target
nucleic acid. In certain embodiments, site specific introduction of
(Sp) or (Rp) phosphorothioate internucleoside linkages into the
central region of a modified oligonucleotide can increase the
therapeutic index of a modified oligonucleotide having a
stereorandom configuration at the phosphorothioate internucleoside
linkages.
[0365] In certain embodiments, site specific introduction of (Sp)
or (Rp) phosphorothioate internucleoside linkages into the central
region of a modified oligonucleotide can alter the protein binding
properties of the modified oligonucleotide. This can also serve to
increase the activity of the modified oligonucleotide and/or
improve the therapeutic index of a modified oligonucleotide. Site
specific introduction of (Sp) or (Rp) phosphorothioate
internucleoside linkages into the central region of a modified
oligonucleotide can weaken the interaction of the modified
oligonucletodie with certain proteins associated with decreased
antisense activity. Site specific introduction of (Sp) or (Rp)
phosphorothioate internucleoside linkages into the central region
of a modified oligonucleotide can increase or strengthen the
interaction of the modified oligonucleotide with certain proteins
associated with increased antisense activity.
[0366] In certain embodiments, specific patterns of (Sp) or (Rp)
phosphorothioate internucleoside linkages into the central region
of a modified oligonucleotide can increase the therapeutic index
compared to a modified oligonucleotide having stereorandom
internucleoside linkages. For example, in certain embodiments,
placement of a single (Rp) phosphorothioate internucleoside linkage
into the 3'-end of the central region of a modified oligonucleotide
can increase the activity of a modified oligonucleotide. In certain
embodiments, having a single (Rp) phosphorothioate internucleoside
linkage in a central region wherein each remaining phosphorothioate
internucleoside linkage has the (Sp) configuration improves the
therapeutic index of the modified oligonucleotide.
Target Nucleic Acids
[0367] In certain embodiments, compounds described herein comprise
or consist of an oligonucleotide that is complementary to a target
nucleic acid. In certain embodiments, the target nucleic acid is an
endogenous RNA molecule. In certain embodiments, the target nucleic
acid encodes a protein. In certain such embodiments, the target
nucleic acid is an mRNA. In certain embodiments, an oligonucleotide
is complementary to both a pre-mRNA and corresponding mRNA but only
the mRNA is the target nucleic acid due to an absence of antisense
activity upon hybridization to the pre-mRNA. In certain
embodiments, an oligonucleotide is complementary to an exon-exon
junction of a target mRNA and is not complementary to the
corresponding pre-mRNA.
Compound Isomers
[0368] Certain compounds described herein (e.g., modified
oligonucleotides) have one or more asymmetric center and thus give
rise to enantiomers, diastereomers, and other stereoisomeric
configurations that may be defined, in terms of absolute
stereochemistry, as (R) or (S), as .alpha. or .beta., such as for
sugar anomers, or as (D) or (L), such as for amino acids, etc.
Compounds provided herein that are drawn or described as having
certain stereoisomeric configurations include only the indicated
compounds. Compounds provided herein that are drawn or described
with undefined stereochemistry include all such possible isomers,
including their stereorandom and optically pure forms. All
tautomeric forms of the compounds provided herein are included
unless otherwise indicated.
[0369] The compounds described herein include variations in which
one or more atoms are replaced with a non-radioactive isotope or
radioactive isotope of the indicated element. For example,
compounds herein that comprise hydrogen atoms encompass all
possible deuterium substitutions for each of the .sup.1H hydrogen
atoms. Isotopic substitutions encompassed by the compounds herein
include but are not limited to: .sup.2H or .sup.3H in place of
.sup.1H, .sup.13C or .sup.14C in place of .sup.15N in place of
.sup.14N, .sup.17O or .sup.18O in place of .sup.16O, and .sup.33S,
.sup.34S, .sup.35S, or .sup.36S in place of .sup.32S. In certain
embodiments, non-radioactive isotopic substitutions may impart new
properties on the oligomeric compound that are beneficial for use
as a therapeutic or research tool. In certain embodiments,
radioactive isotopic substitutions may make the compound suitable
for research or diagnostic purposes such as imaging.
EXAMPLES
Non-limiting Disclosure and Incorporation by Reference
[0370] Although the sequence listing accompanying this filing
identifies each sequence as either "RNA" or "DNA" as required, in
reality, those sequences may be modified with any combination of
chemical modifications. One of skill in the art will readily
appreciate that such designation as "RNA" or "DNA" to describe
modified oligonucleotides is, in certain instances, arbitrary. For
example, an oligonucleotide comprising a nucleoside comprising a
2'-OH sugar moiety and a thymine nucleobase could be described as a
DNA having an RNA sugar, or as an RNA having a DNA nucleobase.
[0371] Accordingly, nucleic acid sequences provided herein,
including, but not limited to those in the sequence listing, are
intended to encompass nucleic acids containing any combination of
unmodified or modified RNA and/or DNA, including, but not limited
to such nucleic acids having modified nucleobases. By way of
further example and without limitation, an oligonucleotide having
the nucleobase sequence "ATCGATCG" encompasses any oligonucleotides
having such nucleobase sequence, whether modified or unmodified,
including, but not limited to, such compounds comprising RNA bases,
such as those having sequence "AUCGAUCG" and those having some DNA
bases and some RNA bases such as "AUCGATCG" and compounds having
other modified nucleobases, such as "AT.sup.mCGAUCG," wherein
.sup.mC indicates a cytosine base comprising a methyl group at the
5-position.
[0372] While certain compounds, compositions and methods described
herein have been described with specificity in accordance with
certain embodiments, the following examples serve only to
illustrate the compounds described herein and are not intended to
limit the same. Each of the references recited in the present
application is incorporated herein by reference in its
entirety.
Example 1 Nucleosides with Chiral Phosphorothioate Linkages
[0373] Modified oligonucleotides containing chirally-controlled
phosphorothioate linkages in the central region were synthesized.
The compounds in the table below are 100% complementary to mouse
CXCL12, GENBANK NT_039353.7 truncated from 69430515 to 69445350
(SEQ ID NO: 1), at position 6877 to 6892. Each compound has the
kkk-d(10)-kkk sugar motif, wherein each "k" represents a
2'-constrained ethyl modified sugar moiety and each "d" represents
a 2'-deoxy sugar moiety. Internucleoside linkages 1, 2, 3, 14, and
15 are stereorandom phosphorothioate linkages. Internucleoside
linkages 4-13 have the stereochemistry indicated in the table
below, wherein a subscript "s" indicates a stereorandom
phosphorothioate internucleoside linkage, a subscript "r" indicates
a phosphorothioate internucleoside linkage having the (Rp)
configuration and a subscript "q" indicates a phosphorothioate
internucleoside linkage having the (Sp) configuration.
##STR00017##
TABLE-US-00001 TABLE 1 Modified oligonucleotides with
stereochemically-controlled phosphorothioate linkages Compound SEQ
ID Chemistry Notation ID NO 558807
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup.-
mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.sub-
.ksA.sub.k 5 1220041
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220042
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220043
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.drT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220044
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.drT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220045
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dr.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220046
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.drT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220051
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dr.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220047
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.drA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220048
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dr.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220049
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.drA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220050
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1237987
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1237988
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.drA.sub.dr.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1237989
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1237990
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.drT.sub.dr.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1237991
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220052
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.drA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220053
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.drA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220054
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.dqT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.drA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220055
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.dqT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.drA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220056
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dq.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.drA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220057
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.dqT.sub.dr.sup.mC.sub.drA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220058
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dq.sup.mC.sub.drA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220059
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mT.sub.dqA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220060
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.drA.sub.dq.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220061
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.drA.sub.dr.sup.mC.sub.dqA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220062
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.drA.sub.dr.sup.mC.sub.drA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 1220063
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.drT.sub.dq.sup-
.mC.sub.dqT.sub.dr.sup.mC.sub.dqA.sub.dq.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1220064
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.dqT.sub.dr.sup-
.mC.sub.drT.sub.dq.sup.mC.sub.drA.sub.dr.sup.mC.sub.dqA.sub.dqT.sub.ksT.su-
b.ksA.sub.k 5 A subscript "d" indicates an unmodified, 2'-deoxy
sugar moiety. A subscript "k" indicates a cEt modified sugar
moiety. A superscript "m" indicates 5-methyl Cytosine. A subscript
"s" indicates a stereorandom phosphorothioate intemucleoside
linkage, a subscript "r" indicates a phosphorothioate
intemucleoside linkage having the (Rp) configuration, and a
subscript "q" indicates a phosphorothioate intemucleoside linkage
having the (Sp) configuration.
[0374] For in vitro activity studies, 3T3-L1 cells were plated at
20,000 cells/well and transfected with 27 nM, 80 nM, 250 nM, 740
nM, 2,222 nM, 6,667 nM, or 20,000 nMnM modified oligonucleotide by
electroporation. mRNA was harvested and analyzed by RT-qPCR. CXCL12
mRNA was detected with primer probe set RTS 2605 (forward sequence
CCAGAGCCAACGTCAAGCAT, SEQ ID NO: 2; reverse sequence:
CAGCCGTGCAACAATCTGAA, SEQ ID NO: 3; probe sequence:
[0375] TGAAAATCCTCAACACTCCAAACTGTGCC, SEQ ID NO: 4) and P21 mRNA
was detected with primer probe set Mm04207341_ml
(ThermoFisher).
[0376] Caspase-3 and caspase-7 activation was measured using the
Caspase-Glo.RTM. 3/7 Assay System (G8090, Promega). Levels of
caspase activation correlate with apoptotic cell death. Results are
presented relative to the caspase activation in control cells not
treated with modified oligonucleotide. Localization of p54nrb in
HeLa cells was visualized with confocal microscopy. HeLa cells were
transfected by lipofectamine 2000 with 200 nM of modified
oligonucleotide for 2 hrs and then cellular protein p54nrb was
stained by mP54 antibody (Santa Cruz Biotech, sc-376865) and DAPI
was used to stain for the nucleus of cells. The number of cells
with nucleolar p54nrb and the total number of cells in the images
were counted. The self-structure Tm of each compound was
determined.
TABLE-US-00002 TABLE 2 In vitro activity, toxicity, and Tm of
modified oligonucleotides complementary to CXCL12 in vitro in vitro
CXCL12 Caspase P21 mRNA % Compound IC.sub.50 (% control) (%
control) nucleolar Tm ID (nM) @ 20 .mu.M @ 20 .mu.M p54nrb
(.degree. C.) 558807 39 1437 353 90 64.4 1220041 388 223 182 0 61.3
1220042 159 584 431 32 62.1 1220043 114 838 488 88 62 1220044 181
489 251 18 61.5 1220045 222 321 259 9.7 61.9 1220046 145 572 635 28
61.7 1220051 237 310 167 20 61.6 1220047 60 814 238 38 61.5 1220048
74 287 174 38 61.3 1220049 77 323 243 17 61.6 1220050 132 174 121
6.4 61.5 1237987 26 317 273 3.9 62.2 1237988 20 336 236 23 62.1
1237989 72 300 394 28 62.2 1237990 186 299 355 14 62.5 1237991 35
562 585 77 63
TABLE-US-00003 TABLE 3 In vitro activity, toxicity, and Tm of
modified oligonucleotides complementary to CXCL12 in vitro in vitro
CXCL12 Caspase P21 mRNA % Compound IC.sub.50 (% control) (%
control) nucleolar ID (nM) @ 20 .mu.M @ 20 .mu.M p54nrb Tm 558807
95 647 235 93 64.4 1220052 63 484 272 98 67.4 1220053 99 621 261 95
66.2 1220054 197 495 192 96 66.8 1220055 51 606 370 100 66.9
1220056 103 569 369 97 67 1220057 104 593 330 92 67.1 1220058 125
578 273 100 67.3 1220059 109 525 351 62 66.7 1220060 61 553 328 100
67.3 1220061 84 409 329 100 67.1 1220062 123 550 394 100 67.1
1220063 111 138 128 12 63.1 1220064 53 160 218 100 65.3
Example 2. Nucleosides with Chiral Phosphorothioate Linkages and
3'-GalNAc
[0377] Modified oligonucleotides containing chirally-controlled
phosphorothioate linkages in the central region and a 3'-GalNAc
were synthesized. The compounds in Table 4 are 100% complementary
to mouse CXCL12, GENBANK NT 039353.7 truncated from 69430515 to
69445350 (SEQ ID NO: 1), at position 6877 to 6892. Each compound
has the kkk-d(10)-kkk sugar motif, wherein each "k" represents a
2'-constrained ethyl modified sugar moiety and each "d" represents
a 2'-deoxy sugar moiety. Internucleoside linkages 1, 2, 3, 14, and
15 are stereorandom phosphorothioate linkages. Internucleoside
linkages 4-13 have the stereochemistry indicated in the table
below, wherein a subscript "s" indicates a stereorandom
phosphorothioate internucleoside linkage, a subscript "r" indicates
a phosphorothioate internucleoside linkage having the (Rp)
configuration and a subscript "q" indicates a phosphorothioate
internucleoside linkage having the (Sp) configuration. [0378]
GalNAc refers to this structure at the 3' end of the molecule:
##STR00018##
TABLE-US-00004 [0378] TABLE 4 Modified oligonucleotides Compound
SEQ ID Chemistry Notation ID NO 558807
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup.-
mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.sub-
.dsA.sub.k 5 855156
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup.-
mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.sub-
.ksA.sub.k-GalNAc 5 1220050
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1277251
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.dqA.sub.dq.sup.mC.sub.dqA.sub.drT.sub.ksT.su-
b.ksA.sub.k-GalNAc 5 1220059
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.dqA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1277252
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.drT.sub.drT.sub.dr.sup-
.mC.sub.drT.sub.dr.sup.mC.sub.dqA.sub.dr.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k-GalNAc 5 1220063
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.drT.sub.dq.sup-
.mC.sub.dqT.sub.dr.sup.mC.sub.dqA.sub.dq.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k 5 1277253
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.drT.sub.dq.sup-
.mC.sub.dqT.sub.dr.sup.mC.sub.dqA.sub.dq.sup.mC.sub.drA.sub.drT.sub.ksT.su-
b.ksA.sub.k-GalNAc 5 1237988
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.drA.sub.dr.sup.mC.sub.dqA.sub.drT.sub.ksA.su-
b.k 5 1277254
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dqG.sub.dqT.sub.dqT.sub.dq.sup-
.mC.sub.dqT.sub.dq.sup.mC.sub.drA.sub.dr.sup.mC.sub.dqA.sub.drT.sub.ksT.su-
b.ksA.sub.k-GalNAc 5 A subscript "d" indicates an unmodified,
2'-deoxy sugar moiety. A subscript "k" indicates a cEt modified
sugar moiety. A superscript "m" indicates 5-methyl Cytosine. A
subscript "s" indicates a stereorandom phosphorothioate
internucleoside linkage, a subscript "r" indicates a
phosphorothioate internucleoside linkage haying the (Rp)
configuration, and a subscript "q" indicates a phosphorothioate
internucleoside linkage haying the (Sp) configuration.
TABLE-US-00005 TABLE 5 In vitro activity and toxicity of modified
oligonucleotides complementary to CXCL12 in vitro in vitro Compound
CXCL12 IC.sub.50 Caspase (% control) % nucleolar ID (nM) @ 20 .mu.M
p54nrb 855156 40 1437 90 1277251 130 174 6.4 1277252 111 525 62
1277253 111 138 12 1277254 20 336 24
Example 3. Nucleosides with Two Chiral Phosphate Linkages in an
Otherwise Stereorandom Phosphorothioate Nucleotide
[0379] Modified oligonucleotides containing chirally-controlled
phosphorothioate linkages at two positions of the central region
were synthesized. The compounds in Table 6 are 100% complementary
to mouse CXCL12, GENBANK NT_039353.7 truncated from 69430515 to
69445350 (SEQ ID NO: 1), at position 6877 to 6892. Each compound
with an ID in the range of 1273959-1273967 has a kkk-d(10)-kkk
sugar motif, wherein each "k" represents a 2'-constrained ethyl
modified sugar moiety and each "d" represents a 2'-deoxy sugar
moiety. Each compound with an ID in the range of 1276491-1276497
has a kkk-d-m-d(8)-kkk sugar motif, wherein each "k" represents a
2'-constrained ethyl modified sugar moiety and each "d" represents
a 2'-deoxy sugar moiety and "m" represents a 2'-Omethyl modified
sugar moiety. Internucleoside linkages are as indicated in the
table below, wherein a subscript "s" indicates a stereorandom
phosphorothioate internucleoside linkage, a subscript "r" indicates
a phosphorothioate internucleoside linkage having the (Rp)
configuration and a subscript "q" indicates a phosphorothioate
internucleoside linkage having the (Sp) configuration. Each
compound contains an "Rp/Sp" unit comprising an internucleoside
linkage having the (Rp) configuration followed by an
internucleoside linkage having the (Sp) configuration, from
5'-3'.
[0380] Compounds were tested in 3T3-L1 cells for caspase activation
as described in Example 1 above.
TABLE-US-00006 TABLE 6 Modified oligonucleotides Compound ID
Chemistry Notation SEQ ID NO 1273959
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.drG.sub.dqT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1273960
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.drT.sub.dqT.sub.ds.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1273961
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.drT.sub.dq.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1273962
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.dr.sup-
.mC.sub.dqT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1273963
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup-
.mC.sub.drT.sub.dq.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1273964
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.dr.sup.mC.sub.dqA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1273965
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.drA.sub.dq.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1273966
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dr.sup.mC.sub.dqA.sub.dsT.sub.ksT.su-
b.ksA.sub.k 5 1273967
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.drA.sub.dqT.sub.ksT.su-
b.dsA.sub.k 5 1276491
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.msT.sub.drT.sub.dq.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1276492
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.msT.sub.dsT.sub.dr.sup-
.mC.sub.dqT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1276493
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.msT.sub.dsT.sub.ds.sup-
.mC.sub.drT.sub.dq.sup.mC.sub.dsA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1276494
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.msT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.dr.sup.mC.sub.dqA.sub.ds.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1276495
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.msT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.drA.sub.dq.sup.mC.sub.dsA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1276496
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.msT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.dr.sup.mC.sub.dqA.sub.dsT.sub.ksT.su-
b.dsA.sub.k 5 1276497
G.sub.ks.sup.mC.sub.ksA.sub.ksT.sub.dsG.sub.msT.sub.dsT.sub.ds.sup-
.mC.sub.dsT.sub.ds.sup.mC.sub.dsA.sub.ds.sup.mC.sub.drA.sub.dqT.sub.ksT.su-
b.dsA.sub.k 5 A subscript "d" indicates an unmodified, 2'-deoxy
sugar moiety. A subscript "k" indicates cEt modified sugar moiety.
A superscript "m" indicates 5-methyl Cytosine. A subscript "s"
indicates a stereorandom phosphorothioate internucleoside linkage,
a subscript "r" indicates a phosphorothioate internucleoside
linkage having the (Rp) configuration, and a subscript "q"
indicates a phosphorothioate internucleoside linkage having the
(Sp) configuration. A subscript "m" represents a 2'-Omethyl
modified sugar moiety.
TABLE-US-00007 TABLE 7 In vitro toxicity of modified
oligonucleotides complementary to CXCL12 Compound in vitro Caspase
ID (% control) @ 20 .mu.M 1273959 1138 1273960 654 1273961 1036
1273962 752 1273963 1349 1273964 907 1273965 984 1273966 750
1273967 785 1276491 116 1276492 450 1276493 234 1276494 85 1276495
214 1276496 165 1276497 148
Sequence CWU 1
1
5114836DNAMus musculus 1cctcccccgt gtctccccac acccgggttg gggttgtttt
ggttgaccag agtggaacac 60aacgatctat tggcagggct gaacaccaat gggtctattt
gtaaagcgcc aatgaccact 120ttctgaagca gggttttagg gagcggggcc
ttagggaact ctttggtcct ttttagaaca 180ctggactttc ttctggaaag
gcaggaaaca ctgaagttta agaagttgtt tccagcttcc 240attaactgaa
cacacattaa aaccaagcac agagaatcag gacgtttcgc gggagtgaga
300cccagtcatt tctcctccgt ttccattctg cagggtgaga gttgtaatca
cccacccact 360attcgtacca tccacccacc cccagtcgag agaatagggg
tacagagggg aggtggcaaa 420gaaaattcac gatactgagt atctctggga
gacctgtttg gtctctttgc tcggtagcgc 480agccctacgt tagaatgcat
cttcccggga atgactgtag tgagactttg gctgggaatc 540caagttattc
taactgtaga ttggtccacg ttgccctaag cctagcagtc cactgcggca
600cagacaccct ggacatgagg tgggtcagct taagttcctg gcacgaaaga
aagggtactc 660tggcaacttt tggatgcggc gaaacagact gtttcgtctc
tcaggttctt atttcacggc 720ttgtgccttt gacagcccct tagtttctct
atctgcagga tgggagcatt aagctctacg 780acccagcctc tttacaattc
aggtccaaag agcccgccca agttggggac tgggaagatc 840aaaggtctca
gcacccagcg gagccgcgga cactgagggc gccaagaagg gggtgggtag
900gtagggaact ggaagggcgg ctgctccgca ggggatgcgc gtcagagacc
ccagccacac 960tccaggcccg ccccttgatg agccccgccc cgccccgcct
ggttttcgcc tctaaagcgc 1020ccagcgctcg cctcccgctg ccgcactttc
actctcggtc cacctcggtg tcctcttgct 1080gtccagctct gcagcctccg
gcgcgccctc ccgcccacgc catggacgcc aaggtcgtcg 1140ccgtgctggc
cctggtgctg gccgcgctct gcatcagtga cggtgagtgc aatccgcggc
1200cgggcccggg aaaggctcgc agctctgcgc cggagctcct tcgggtccgc
ggttcctctg 1260cccgcgccga agtcgcggag aaagaactcg gtcggcgccg
ttcactacaa gcgaacttgg 1320ggcagtccac tttgcagggc gcactcccac
cgggtgccct ttcccgtgtc ccacgggtcg 1380caccgaggtt ttgtgctctg
cgaagtgcgg ccataggacc tagagagggc tgcaggggag 1440gacccgcagg
attgttgggc aagagtgggt tcggcgcgga atggaagcgt gggcgattgt
1500gtccggggct tgggccccgg agcgcgccag ctgcactcag ctagtgtcta
ccggcgccca 1560gatgtttcca gaggcgaagg gcagcgcggt cccggagttg
accgtgcaag aggttcactc 1620gggtggtgcg tgtgtcagca aactctcaaa
gaccggtcaa gtagctcgaa gtgcatggct 1680tggctatagg ttcagtggtg
aggctgagtt tcgtcccctg cgggtgtagc gtgttctctt 1740acagcaccct
cgaggggctc agggccacca gcagcgcagc gcagctcttg aactcgcgct
1800gccagccagg gccgcgcttc tgcacagttc gttggtccgt agcgacgcgg
acctgagcac 1860gcgtctcttc actgcccctt tttcttctta cccgggtcac
tagacaaagg ctcagcagtt 1920acccaagcta tatgcacacc tctccccaac
ccccaaacac acctgcaaac gggcgctttt 1980gtagccagcc ccggagtcct
cagctctgga atgagagctg cagcggagtt cagtctccca 2040gacccagggt
ggtgtcttct ttcactggga aagggctttc attttgtttt ctttttttga
2100cactgaagag aaaactctca gcgctgttac aagacaccgt tgctgcaaaa
caaaacaaac 2160cattgcctct gaacacaaaa caaaatccta ctagtcgatc
ccctgccttc ctccgcagtg 2220gtgtttcctg gagagaactg agggacagtc
ggggctcttg gtgagactga gctctaaatg 2280ctgcccaagt acaccaactc
gttcgtttgg gttctttccc tgtgacaacg gggtacggga 2340atggttggag
ttgcctagtc cgagggaaat gttctgtaaa agaatagtca gttgctgatc
2400ggagtagtaa aaaaaaagaa atgaaaggca gtttcgattt tttttttttt
tttttttttt 2460ttttttgtta ccgagaacac ccgggaggct gagccttccc
actggtcccc cagtgccccg 2520tcatggagca cattgatttg ggcattaata
attgaatgag ctggtgatgt tgcaagggtc 2580acagcctctg gcaagttagg
tatggggcaa gaatgtagga ctcaggtcct caaggttgga 2640gtgcaattat
ccagagtaaa agttgtctca ccctcaacat attctgaccc taggaagagt
2700cggattgttg acagtgtctg gatcagacct gttctctagg caggacccca
ttgtgctgcc 2760cgaatgaact tttttacctc ctagtgcctg tgtgccctct
gatcttacac agccctcaag 2820ttgcagcacg gctaaccttg ctgtggttcc
tgtcttttcc catcagctac tccaactcag 2880aagctagata gtagacaccg
gaggcttctt tggttaaacc cagagcagca ggcttgccag 2940gcttgttaga
ttgaatggac ccctggttcc ctaagccaag ctctctagat tcccaagtcc
3000agggtggcag cagagctgga ttagactttg gtctgtacct gaagtctggt
tttcctatgc 3060tttagagtct aaagacacta cccttcctgg ggcatgcatc
ccttagctaa ataatgcttg 3120cagaagaaga taatcccatc atatatttaa
ttcggtccac ttctccagct gcttcccaaa 3180ggcagtgaac ttcagaatac
ccagaagtct cctggaactc taaataagca aacttaaaat 3240cctggggcta
actattctca gtcatacttt taaactttgg tgaaaagacc cataaattga
3300aacatttggg gatgctcagt agagctagga taaaaccctg ttgttggggg
agcagctaca 3360aatccagcag tcctcagggt ttgcaattct agacttaaag
ggtggttctt aagggggggt 3420tctaaaggag ccccttgcta atttacacta
atgagtgtca attatagcat tttgcaaatt 3480ggtgaattgg caaacaaagc
tggtaatagg atccaggagg cctaggcatc caggtagtga 3540ccataaaagc
cacggttgac cccagctttt gggaaaagct ggatagaagg taaatccggg
3600tcctcccctc tggattcttt tgtgatttcc agggcttagg atagggtgag
tgggaggagg 3660gaaaactgca ggtggtagaa gtgaagcccc ccacctccag
gcctgcacca gagggccaca 3720agggagccca gaactctgcc accccacttc
tcctgggtcc ttttgtcctt tagaggctga 3780gcccagtcag atctcactgt
gatccctggc cgaggggatg gtctttgcaa gaaactttct 3840gtaaccattc
ctgctgatgt tcctgagtct tccccacaag agccaccaaa ccccctgcac
3900caggcagata atgactggcc ccacttttct ctctacacct cctctaggta
aaccagtcag 3960cctgagctac cgatgcccct gccggttctt cgagagccac
atcgccagag ccaacgtcaa 4020gcatctgaaa atcctcaaca ctccaaactg
tgcccttcag attgtgtaag tcctagccgc 4080catcccccaa agaggagcat
ggtatagaag cctcggactt ggcataacta ggggcagctg 4140ttaccaccac
caccacgggg acactgatat gccatcagac atgggtttca aaggatactt
4200ttgttcccca gagccctgat gtcctcagtg tttctcactc ttgctttcca
agctgtttct 4260tgcagcacag tgggccgcct ctctacagaa aaagccatgg
acttgatgga ggtcagccct 4320cagctgacag ttgggtctgt cttgtcagtt
tcaaggttct ggtgtccaaa gttaatcctt 4380tctcacatag aaaaaaaaat
tacaagaccc ggatggcacg gggggggggg gggttcagtt 4440ttactcactt
gcactcactt gctcagaggt catttttgtt ttagagtttt agagtttgct
4500ggagtgtgat ggtagctgcc agtatttgat ttaaatttac ctgggaaata
agaaaagccc 4560aaaaaaggta taaatgatgt gaatatctca ctcagagtct
ggtagacttg gcagagatgt 4620gtcctgtgct agtctgtcct gctcactgcc
ccccagcagg ggttcccatc ctcgggagac 4680tcaacactaa caacagtata
aggatgcagc agctggagca atgctagcct gacggctttg 4740tcacccaacg
gtgactgctt cagactttct gtgctcatca gccttcctct ccagcctccg
4800ctgctgtgtt atgtacagta ggctttagag acctagatga tgaatattat
ttttgctgtt 4860ttgattaaaa tacaatactc tcccgagaaa gggattttaa
agatgatgag tttacgtttg 4920aataggctgt gctggtgcac tgtcccggga
agggcccttg aacttagagg gtcaaataca 4980actattgatt ctgggtgatc
actaagttaa taaatggcag gatccagact gacacccctg 5040atccctgttg
aagttacatc cctctgaacg actggtcaac tgcagggcag cctgcttgaa
5100gagggttacc tgtccctagg acactgaaca ggcatttgtt tttcctagaa
gacagttcac 5160cagctggaga ggagtcgtct cccgtagttt ctgtttggtt
gcttttggtt tttgtttggt 5220tttggttttt taattatctg gcatccagga
cttgatggaa aataaccaga gctaagctca 5280ccggttcatc tgcccattag
gaagttctag ggatgggaga aagaacacgg cgtcaattaa 5340caaatccaca
aagctaagac cttgaagcat tctgtgaact tgtaaacgcg ctcaggcaac
5400cattggacaa tttgtctaga ctgctccttg cccacctgaa ctgccctgtt
cctccccttc 5460tggactcctg ccgtcttcct ccagagctac ctttaaggtt
gtcccatgta ctatcaaggt 5520gctctgtcaa aagttcttag gctgcttctg
gcactctcca gaattttcca agacctcccc 5580cccaccatga tatcagtcat
ccgcgccttc tgggtggttc ttcctccaca ccctttgggc 5640actttgactc
ctgtgggata ttcgtccttc cttttccttt agctttcctc acttgccaag
5700ctccaacttg gccagaagct caaatgcctc cactgtggtc tcttctctgt
gtcccctggg 5760agacatcctt agcacgtccc taactctgcg gtggtggtcc
caacacgatt caagtgctat 5820gtcttccaaa actgaagctt ccgggagcag
cagctgggcc ctgcagtgag gacctttagc 5880tgggtgtgtt gggtgagccc
acaggatcgc tttctcccgc ttggctgtac agcgtctctc 5940cccttgtgtt
ttggcagtgc acggctgaag aacaacaaca gacaagtgtg cattgacccg
6000aaattaaagt ggatccaaga gtacctggag aaagctttaa acaagtaagc
acaacagccc 6060aaaggacttt ccagtagacc cccgaggaag gctgacatcc
gtgggagatg caagggcagt 6120ggtggggagg agggcctgaa ccctggccag
gatggccggc gggacagcac tgactggggt 6180catgctaagg tttgccagca
taaagacact ccgccatagc atatggtacg atattgcagc 6240ttatattcat
ccctgccctc gcccgtgcac aatggagctt ttataactgg ggtttttcta
6300aggaattgta ttaccctaac cagttagctt catccccatt ctcctcatcc
tcatcttcat 6360tttaaaaagc agtgattact tcaagggctg tattcagttt
gctttggagc ttctctttgc 6420cctggggcct ctgggcacag ttatagacgg
tggctttgca gggagcccta gagagaaacc 6480ttccaccaga gcagagtccg
aggaacgctg cagggcttgt cctgcagggg gcgctcctcg 6540acagatgcct
tgtcctgagt caacacaaga tccggcagag ggaggctcct ttatccagtt
6600cagtgccagg gtcgggaagc ttcctttaga agtgatccct gaagctgtgc
tcagagaccc 6660tttcctagcc gttcctgctc tctgcttgcc tccaaacgca
tgcttcatct gacttccgct 6720tctcacctct gtagcctgac ggaccaatgc
tgcaatggaa gggaggagag tgatgtgggg 6780tgccccctcc ctctcttccc
tttgctttcc tctcacttgg gccctttgtg agatttttct 6840ttggcctcct
gtagaatgga gccagaccat cctggataat gtgagaacat gcctagattt
6900acccacaaaa cacaagtctg agaattaatc ataaacggaa gtttaaatga
ggatttggac 6960tttggtaatt gtccctgagt cctatatatt tcaacagtgg
ctctatgggc tctgatcgaa 7020tatcagtgat gaaaataata ataataataa
taataacgaa taagccagaa tcttgccatg 7080aagccacagt ggggattctg
ggttccaatc agaaatggag acaagataaa acttgcatac 7140attcttatga
tcacagacgg ccctggtggt ttttggtaac tatttacaag gcattttttt
7200acatatattt ttgtgcactt tttatgtttc tttggaagac aaatgtattt
cagaatatat 7260ttgtagtcaa ttcatatatt tgaagtggag ccatagtaat
gccagtagat atctctatga 7320tcttgagcta ctggcaactt gtaaagaaat
atatatgaca tataaatgta ttgtagcttt 7380ccggtgtcag ccacggtgta
tttttccact tggaatgaaa ttgtatcaac tgtgacatta 7440tatgcactag
caataaaatg ctaattgttt catgctgtaa acctcctacc gtatgtggga
7500atttatttac ctgaaataaa atctactagt tgttagatgg agtgcacata
catttctgaa 7560gatggagaaa aacaggtgtg cctgctgatc aggtgctgtg
ggctgccctg cagtcctggt 7620gagcgacaga cactgaggca ggcttgtctc
atgaacaggc tgcctctgca gtgaaagttt 7680ttgtgtattt tttttaaccc
aagctagttt tctaatgaat aatacttgac tcactaattt 7740cccctcctcc
tccttctcct cagttctcct aacatcctca tgtgatcccc agactcaact
7800ccagtaatat caagctttcc tattttccca tgtaaaaaaa tcccatgact
ctgggccatg 7860ttaatatcag gcttttgtgg gaacaggtgg cctcacccca
taaatcatta aataccattc 7920agcttgaatc attttaatgt gacagtcaca
aaccagttgc tctaataaaa actctgctaa 7980ccatccttct ccttagctct
ctagaacaat ctcagttatc cctagggatg ctccccagca 8040tccagaaaag
agaagtggga tcaatcatcc tgcctttctc cccctcctct cttggagggc
8100tgcctgagcc cgtggcctcc acctcccctg ctttgtataa tttgaaatgc
agatttgtag 8160tgaaggcaga gttcacctct gcattgaaag ggaaggcagg
cccagagctt ccttccctgc 8220cctctgagat gtgcatttat gtctcaggat
ggatgagctt tggtaggaat gctcaaaacc 8280aggaccagcc agacaaactg
gcagtccctg taagcggttc ccgggtcata gggttagggc 8340acccctgttt
aactttgggg tggggaaagt atctggtttt ctttgataaa ttgcttgtga
8400accacatttg ccaagtggcc tccaggcctc aaactcaaag accgagctaa
atcgactcgg 8460aaggcaatgc tgaatgaaga ttgtgggaac tgagatagat
acactcctct atgttgcaat 8520gtgattaatg gttctactaa ttttatctaa
gggggcgcag agaagaaaaa gtggggaaaa 8580aagaaaagat aggaaaaaag
aagcgacaga agaagagaaa ggctgcccag aaaaggaaaa 8640actagttccc
cgcttcctgc cgatggaccg cagtgcgctc tgctctggcg ctttgtaact
8700cgctcctccc tcttcggggg cagaccccac actccgggca ggtgctcaaa
cctgacggta 8760aactcttccc tcttcggggg cagaccccat accccggggc
gggtgcttag gctttcctgc 8820cctggtggcc acaccagctg ctgtatttat
gtgcttcata aggccctgct ctgtctgcta 8880aagctatgaa gaaagatgtg
cagagactgg ggtggagact aagccaaaga ggagctgcct 8940agcctggcag
cattgccccg agctgagccc ccttggccag gacttcacaa ggctcacacc
9000tacaatccca tgaaggccag ggtggtctgc ttagccagga aagggcaagt
gccttcccct 9060cggccacact gccccttgtg gccttctcgg gacatgtggt
aactgacttg ctctcaggcc 9120cacccgcagc ttttccaaat acctgcagcc
ttcagccctg ctgccctgcc tgtgggagca 9180gctttgactc cagtccagaa
gggtttctgc agactgtgtt gggtgagacg cagaaaggat 9240gaaatctcag
aacacatgtc agctgcttct caggaaatct tttctttgga caattcactt
9300tagagtcttt aaacgggtct ctcgtgggga ggatagatgt gctctggaac
tttctgaagg 9360accagcagct tcagggactc ttagtctgtc cttccccact
tttggtccca acatccctgg 9420gatggtgtgc tgtctgggca ccacggtctc
catcctcact cctgagagat ttctgccttc 9480tgtgagttgg gttaaagctc
tggaattatc tactatccca atccactacc ctcacctggc 9540aatatttgtc
tgtttttgtt tgtttgtttg tttgtttttg tcttttgcca gtttgaatta
9600gaaggcaagg ctctgatttt agtagtgttt tggaaaagga cttttttctt
caccttcctc 9660tttgcctcat gtgtacacac acacacacat cttgtacccc
agacctctgg gtataatttt 9720cataattggt gcagaaagaa gaaatgatct
gaagatgtgt taaatggatt gcaggggaag 9780gaaggcccag ggccctgtgt
gtcatgccct cttgggttcc taagttctat gttccttaga 9840ggttctagca
ttaaacagat aaagcccttc atggtcctgg ctgaggaaga gtcttgctag
9900ggggattcag ggaagacccg tgttaccagc tcttaccctt tatctggaca
gctctcctac 9960cctgtatctt ctcctcagat ctgaggatag caggctggac
tattggtggg cacctttcaa 10020gcccagggct actgtttgtc ctgtggcagc
cggctacagt ctcgtctgag tggcctcatc 10080tggacccttc ctgttattaa
taaaacgctt ctggaggcca gatctgtgct caagccatag 10140ttctgcttag
aaagggatgc cccaccctta ccggacactg ggaagaactg ttggccccta
10200gaaaccaaag gccaaactga ggctgccctg agttggaaga ccactttctg
aaatgcccat 10260ggactctgcc tcccaaccat tcgtctctca ctcctagcag
agctgtctgt gcagactgtt 10320tcttaggagg cacagcaagc tccagggaac
cctctgtgct tatgaagctc gtctggtggg 10380caaccccagc ccactggaca
gagtcctcat ggaaatgcct gggaagctga tttcatctaa 10440ggatgggttg
aagtaggatg tgctcctgcg acttctcagg caggtgagag gggtagtcct
10500tacactgtct agcataaacg ccttccggaa ggacctgcag ctccagagac
cacctcctga 10560gcaccaagac ctcttctggt ggtgtggaac cagccaagag
atttcaagga agagtgatta 10620tttgatgaat gctatgggaa tggcctcttc
tcttggagtt ctgaggcctg gggatgccca 10680ggaacactgg gcacctgctg
ctgttagggc caatgcatag tctcagcacc ggtgtcctaa 10740ggttaaggcg
gtgcgccttg tcatgtgctc cttgtaccat gccatctgtg ccagtgtgtg
10800tctgcctcac cctgtgcttg acatgttcac ccatcttctc tgcttcccgc
caccatccag 10860atcctcagcg gccgccccgg ctgtgccctt ccctgctctc
ccgctctctc aggcctcgga 10920aggaagatcg gtggctgcga gctgaactaa
ggagtagggc ctgtggctca gcgctaggcc 10980acgcacgcag catcccaggc
atgtggtgag aaactgcctt aatgtgtctc ctctgttctt 11040gtcaacagga
ggctcaagat gtgagaggtg tgagtcagac gcccgaggaa cttacaggag
11100gagcctaggt ctgaagtcag tgttagggaa gggcccatag ccacttcctc
tgctcctgag 11160cagggctgaa gccgtttgca agggacttgc tttgcacagt
tttgctgtac tttcacattt 11220tattatgtag caagatacat ggtgattttt
ttttttttca tttagcctga ttttccaacg 11280tcattggtga caggccaagg
ccactatgtt atttcctttg ttctggtatc cttcccttgg 11340aggaccttct
ctgagtagtg gctccccagg tttgtccttt gagctgaggc aggaggctca
11400cccattcttc tgaataggaa ctgggtgttc ccacccccca aggactgcag
ggctttccca 11460agctgaggca ggaacgtgag gccagggaag agtgagcttc
accctcatcc cacgctgtcc 11520tcctcaaccc accatgctca tcattctgtc
tcatccatcc atccatccat ccattcatcg 11580ccatgtgtcc gcaagactgt
ctccatgacc ctgaaaaagg actctcgaga tgaaatcctt 11640tattcaaatg
ggacagcaag aaggaaaagc caatgtctgg tgtctctccc cccgccccta
11700ccctgcgcgc atctatgtct tgtttggaat attgtctctt caaccccctg
ttcatgtcct 11760tctcactcat gatcgatgtc ttgtctgtgc actgtctcta
acccaaatgc aaaggctgag 11820tgtgaggtga tggccccgag gtccaggttg
tagtcatgga aagagccctg ctgtctccct 11880tctcaggggg cccattttag
acacacaaag cccaaagaaa ggtggtttgc aacagtgctt 11940agctcgagcc
tccatatttc cataactgtt agcttaaaac tgtggggttt taccttcctg
12000gaaccaaatg cattcttctg ttgaggagta acaggtctca attcttttca
attaatttta 12060aaagtcaatc actaagagca tcggctttgg gccctgatgg
gcaggcattt ccctggaaag 12120ggggtgaact acctacctct cctcaagaca
gccgaagggt gggattggtg ccgctctggg 12180aagcgtggcc ccaggagttt
tgtcctctgc agtttttaat gcaagttcac tgccactttg 12240acaaaagccc
aattagaagc cagtctctag ttccttaaac aaaacagaca gagtaaggaa
12300aggaaggagg gtggcagcca gctggctgga cactcgagaa agacggggaa
gtaagctaca 12360gaaagatagt cttcaaaaac aggtgtttga gagtgaatac
tctgtagaat tgttagtggg 12420gtgtgtgtgg tggtggtggg gggatttcta
caaaatagtc ctttaagttg agtttacagc 12480agatgaaaaa tccaaccagc
aaaattttga tcaaatttga acaaaaaccc aaaaacctaa 12540aactgttgag
caggttgcga tgaggagcac agggctagct gcagagctgg atcctcagga
12600ggatagcgaa ttattttcaa ccctggaata gaaaccacac actggcttgc
tgtgcaccag 12660ccactttgca tctaatccaa gctttgaagg gtgttgcttg
ggaggaaaca aatacagcct 12720tccatcttca ctccagttag ggatcctttc
aaagtctcct tcacagtgag gaaaaagaga 12780agggtagaaa ctttagggag
ccggatttgt gtatcaattc ctccgctgac agtcagtttc 12840tagatggaga
cagcctgctt aaagcaaatc cgaatttaaa taggacattt acatcggaaa
12900agtctctccc taccttaatc ccccattctc ttgctttcaa aatacaagca
cagcagtcct 12960tgaatggctg ttgacccagg gcacctagct gtccctgctg
gtcctggggc tgccagaatt 13020cccttgggcg ccaagcaacc tgccaggtag
ccagtccctc tgttacaagc ctttgcatct 13080ggatagggaa aggggtggag
acatacagtc tgctttgtgt tgaaacccag atttgtaccc 13140tgtgtttata
cactgctgct ggctcccgag gacagtggga ctttagcaag gaagtgcagc
13200cgaggggtaa agagccctct ggttcattgc ctgatcggct ttgagagagg
gtttggaggg 13260caaggggctg cattcctctg agggacttgg cctgaggcct
ttcgggcctc tccagtgggt 13320tctgtttatc ctctcatggg tgattatctc
agtggtgtca ccaggggctt cctcccagaa 13380gtcagtcatc cccaggccgt
gcaccctttt cagctggatg agagccaggg atgcattctc 13440tccaaacagc
taccctggcc cattttaagg taatctcatt cttcaaaatg ttccatagaa
13500tcctccaaat tcccccagca gacttctacc ctcgccaagt tcccaaaacc
cactcagcaa 13560agttgccaac ctcgacgggc tagcagtgtc taagcagcga
tgggttcagt gttgtgtgtg 13620gtgaatactg tattttgttt cagttctgtc
tcccagataa tgtgaaaacg gtccaggaga 13680aggcagcttc ctatatgcag
cgtgtgcttt cttattctta tttttaatat atgacagtta 13740tttgagaagc
catttctact ttgaagtcat tatcgatgaa agtgatgtat cttcacctac
13800cattttccta ataaagttct gtattcaaat atagctgcca agcatcctca
gtgaatgtta 13860ccatgtggaa ttttccacac ttggttttac cccctcaaac
ctgactctga ccgtgcagtc 13920ttagcagaag agcttagcag gtcctagtgt
tcactcttgg tctaactgct ggtgtcagaa 13980gatctctaca gggagaggtg
ttccattttc tccacatgac ctggattgct ccttagaggt 14040cagacagcct
tgcactgtac aaggcaatgg cttagggtaa agtcccagga gttttcccta
14100cagtcccaag aatttggaag aggaaggccc acactacaca tgcaggtcat
ggtggaaggt 14160gacagaggaa ggactctgtc cctgtaagac agctggaaac
cacaatattc tgcatgttcc 14220tatcctgggt gaggacgcta atggaagtca
aaggggaatt tgctaactgc tgttggccag 14280cttcctccaa gaatcctgct
tccccaacag acagagcctt tgtctcttat agtttggtct 14340tcagattctc
tttatcccac attcagccat ttttgtaaaa gagaggctag caccagctcc
14400aaatatccaa atctgcagtg tttgagatct cactgcgcct cctccatacc
aacacatttg 14460ccattactta tagggtagtt ttcatgtgag ttctaagttg
attaacacac aagaattaga 14520agggtgggag gctctaggaa aggcactgtg
ggactatttg actgcatggg tgtgaaaatg 14580taaggaacag gcaagagctt
ggatcccatt ctctctgccc acattgtgac ttgagatata 14640ctaattgctc
ttgggggtct cagtcatata ccatccataa cagagttaaa ctgagagaga
14700tacaggatca gctagaatga aaagcccacc ccatgcttcc agaaagtccc
ctctttatac 14760ctcctgtgat atgaactaga ggaaaagcaa ttgactttgc
ttctcaaaca gcctacggca 14820aagccctgtg agtttg 14836220DNAArtificial
sequencePrimer 2ccagagccaa cgtcaagcat 20320DNAArtificial
sequencePrimer 3cagccgtgca acaatctgaa
20429DNAArtificial sequenceProbe 4tgaaaatcct caacactcca aactgtgcc
29516DNAArtificial sequenceSynthetic oligonucleotide 5gcatgttctc
acatta 16
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